Detecting microbial infections in wounds

ABSTRACT

Provided herein are microbial infection indicator devices, including dressing with indicators, standalone indicator inserts or disks that can be freely placed at a wound site or dressing, and applications thereof for displaying a visible or detectable signal to a user upon detection of an analyte or biomarker indicative of an infection, such as a color change.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of United States ProvisionalApplication No. 62/315,565, filed Mar. 30, 2016, the disclosure in whichis incorporated herein by reference in its entirety and made a parthereof.

TECHNICAL FIELD

Embodiments described herein generally relate to wound healing, and inparticular to compositions, apparatuses and methods for the detectionand treatment of wounds.

BACKGROUND

In mammals, dermal injury triggers an organized complex cascade ofcellular and biochemical events that result in a healed wound. Woundhealing is a complex dynamic process that results in the restoration ofanatomic continuity and function: an ideally healed wound is one thathas returned to normal anatomic structure, function, and appearance. Atypical wound heals via a model consisting of four stages—‘exudative’phase, proliferative phase, reparative phase and epithelial maturation(Hatz et al., Wound Healing and Wound Management, Springer-Verlag,Munich, 1994) or hemostatic, inflammatory, proliferative and remodelingphase (Nwomeh et al., Clin. Plast. Surg. 1998, 25, 341). Theinflammatory phase is particularly important to the wound healingprocess, wherein biochemical reactions at the wound situs facilitatehealing but also cause tissue breakdown due to production of excessproteases.

Infection of the wound results in either a slower, or an arrestedhealing process. For example, pathogens in a wound can produce toxins(e.g., Clostridium species), generate noxious metabolites like ammoniathat raise pH (e.g., Proteus species), activate or produce tissue lyticenzymes like proteases, or promote tissue invasion, thereby leading toan increase in the size or seriousness of the wound. In a worst case,pathogens can leave the wound and cause sepsis.

In order to keep the chronicity of wounds in check, a variety ofassessment techniques and/or tools are employed in the clinical andveterinary setting. Current methods of assessing an infected wound arebased primarily on assaying for a variety of parameters associated withthe wound. For instance, a wound may be assessed visually, length anddepth measurements may be taken, digital photography may be used whereavailable to track the visual condition and size of a wound (Krasner etal., supra). In clinical practice, diagnosis of infection is based onmeasurement of secondary parameters, such as, odor, presence of localpain, heat, swelling, discharge, and redness. Many of these clinicalindicators, such as inflammation and discharge have a low predictivevalue of infection in wounds. In other instances, the number(s) andtype(s) of pathogenic flora at the wound situs may be determined usinglaboratory and/or clinical diagnostic procedures. Swabbing of a woundfollowed by microbiology testing in the hospital laboratory is an optionfor confirmation of bacterial colonization and identification of thestrains associated with infection, thus allowing for the prescription ofcorrect antibiotic course. However, this process is time consuming andlabor intensive. Delay in diagnosis of infection can delay theadministration of antibiotics and may increase the risk of developingsepsis.

One of the biggest drawbacks associated with existing clinicaldiagnostics is a lag associated with the onset of infection and thetiming of detection. For instance, positive identification of infectionusing swabbing procedures often depends on attainment of a “criticalmass” of microorganisms at the wound site and so early detection cannotbe made until a detectable level is reached. Also, the swabs may becontaminated with the flora of the surrounding tissue, therebycomplicating the diagnostic procedure. Other drawbacks include, e.g.,sampling errors, delays in transport of the swabs, errors in analyticalprocedures, and/or errors in reporting. See, the review by Bowler etal., Clin Microbiol Rev. 14(2): 244-269, 2001.

There is therefore an imminent but unmet need for diagnostic reagentsand methods that enable early diagnosis of clinical infection,preferably, which permit clinical diagnosis prior to manifestation ofclinical symptoms of infection. There is also a need for compositionsand methods that would assist in predicting clinical infection of awound prior to the manifestation of clinical symptoms. Such a prognosticaid would allow early intervention with suitable treatment (e.g.,antimicrobial treatment) before the wound is exacerbated and surgery orother drastic intervention is required to prevent further infection.Additionally, if clinicians could respond to wound infection as early aspossible, the infection could also be treated with minimal antibioticusage. This would reduce the need for hospitalization and would reducethe risk of secondary infections, e.g., as a result of contact withother diseased subjects.

SUMMARY

The technology disclosed herein provides for compositions and methods ofdetecting infected and/or chronic wounds. The disclosed technologyimproves upon exiting assays by: increasing the sensitivity, precisionand specificity of detection of infected wounds; providing for theability of qualitative and quantitative measurements; and, increasingthe speed of detection of infected wounds in situ and in real-time. Theassays and methods described herein are partly based on the use ofspecific reagents that detect biomarkers and/or probes which are presentin infected or chronic wounds. The detection process may involve use ofreagents that are specific to the markers present in infected wounds butnot non-infected or non-chronic wounds and the detection step mayinvolve qualitative or quantitative measurements of the signal(s) thatare generated when the probe is acted upon by the marker. In embodimentswherein the detection method involves detection of enzymes present inwounds, the probes comprise modified enzyme substrates that are specificto the enzyme, which generate signals that may be optionally amplified.This greatly improves efficiency and specificity of detection. Moreover,a plurality of detection probes, each specific for one or more targets,e.g., enzymes that are specific to the wounds, may be employed. Thisgreatly helps to maximize both efficiency and accuracy of diagnosticassays while minimizing the incidence of false positives (e.g., duenon-specific interactions and/or target redundancy). Furthermore, theexperimental results disclosed herein confirm that the novel probes andthe assay techniques based thereon are capable of detecting andcharacterizing various types of wounds. Finally, the reagents of thedisclosed technology may be used together with therapeutic moleculessuch as antibiotics, antifungal agents, etc. to monitor and evaluatetreatment and management of chronic wounds.

Embodiments described herein are based, in part, on the discovery thatcells of the immune system, including enzymes generated thereby, mayserve as markers in the early diagnosis of wounds. These cells, e.g.,neutrophils, are recruited at the wound situs to combat infection, do soby engulfing bacteria (and other pathogens) and/or neutralizing themwith enzymes. Some enzymes are specific towards proteins (e.g.,elastase, cathepsin G), others are specific towards cell wall components(e.g., lysozyme) and yet others mediate protein denaturation (e.g.,NADPH oxidase, xanthine oxidase, myeloperoxidase (MPO) and otherperoxidases). These cells, e.g., neutrophils, are generally onlyshort-lived and when they lyse in the area of the infection, theyrelease the contents of their lysosomes including the enzymes, which canthen be detected to provide a reliable measurement of the status of thewound.

Accordingly, various embodiments described herein utilize the detectionof enzyme markers, which are indicative of the presence of myeloidcells, and neutrophils in particular, in a biological sample ofinterest, for example, wound tissue. Increased level or activity of suchenzymes in the wound fluid, therefore, corresponds to a heightenedbacterial challenge and a manifestation of disturbed host/bacteriaequilibrium in favor of the invasive bacteria.

Provided herein are embodiments of a wound dressing, devices, andmethods for detecting an infection in a wound or a sample. Oneembodiment is a wound dressing comprising a wound contacting layer, areagent layer comprising one or more testing regions, wherein thereagent layer is in fluid communication with the wound contacting layer,and an outer layer that overlays the reagent layer. In some embodiments,the wound contacting layer comprises gel-forming polymers. In furtherembodiments, each of the one or more testing regions comprises one ormore of each of: back-flow trap, reagent pad, filter pad, indicatortrap, and absorbent area, wherein one or more viewing windows arelocated either above the reagent pad or the indicator trap. In furtherembodiments, the reagent pad is in fluid communication with the filterpad; the filter pad is in fluid communication with the indicator trap;and the indicator trap is in fluid communication with the absorbentarea.

In other embodiments, one or more testing regions comprises one or morereagents selected from the group consisting of enzyme-reactiveindicators, reagents that are sources of peroxide, enzymes that producecolored products, pH indicators, protein responsive reagents, andmoisture-detecting reagents. The enzyme-reactive indicators includeprotein-indicator conjugates printed, sprayed, or otherwise deposited inor on the reagent pad. In some embodiments, the protein-indicatorconjugate has the structure of Formula (I): A-B, wherein A is an anchorregion or moiety that helps to bind an enzyme-reactive region to thereagent pad, and B is the enzyme-reactive region.

In some embodiments, the enzyme-reactive region comprises a peptideand/or an indicator region. In further embodiments, the wound dressingcomprises an indicator region that after having been cleaved by thetarget enzyme in a sample is further transformed into a colored speciesby accessory enzymes selected from a lipase, esterase, hexosaminidase,peroxidase, oxidase, galactosidase, glycosidase, glucosidase, andlaccase, or a combination of two or more thereof. In some embodiments,the enzyme-reactive indicators interact with elastase, lysozyme,cathepsin G, myeloperoxidase, or any combination thereof. In furtherembodiments, the enzyme-reactive indicators comprise a moiety capable ofproducing a visible color or a detectable electronic change uponinteraction of the enzyme-labile or enzyme-reactive region with one ormore enzymes, wherein the moiety is selected from the group consistingof a peroxidase substrate, arylamine, an amino phenol, a neutral dye, acharged dye, a nanoparticle, a colloidal gold particle, or an analogthereof. The anchor region can be attached to the reagent padcovalently, non-covalently, or ionically. In some embodiments,pH-sensitive reagents produce a visible color comprise bromothymol blue,phenol red, bromophenol red, chlorophenol red, thymol blue, bromocresolgreen, bromocresol purple; nitrazine yellow; or other sulfophthaleindyes.

In some embodiments, the wound dressing also comprises one or more linesof wicking stitching or wicking tufting throughout all layers of thewound dressing except the outer layer, wherein the wicking stitching orwicking tufting provides fluid communication between the reagent layerand the wound contacting layer. Fibers that are wettable and exhibitcapillary action may be used for wicking stitching or wicking tufting toform fluid communication between a sample or a wound and the reagents.In some embodiments, the wicking fibers are solid or hollow. Examples ofwicking fibers include, but are not limited to, cotton, rayon, viscose,wool, silk, polyester, polyamide, CMC, and polypropylene.

In further embodiments, the wound dressing comprises one or more testingregions, comprising a leach-back trap in fluid communication with thereagent pad and one or more lines of wicking stitching or wickingtufting crossing through one or more testing regions only at theleach-back trap. In some embodiments, a foam layer is added between thewound contacting layer and the reagent layer. One or more perforationscan be added in the wound contacting layer or in the foam layer and thewound contacting layer. In further embodiments, each of the one or moretesting regions further comprises a leach-back trap in fluidcommunication with the reagent pad and one or more perforations alignedwith the leach-back trap.

In some embodiments, the testing regions comprise a multichannel testingregion, wherein each channel within the multichannel testing region isseparated from an adjacent channel by one or more impermeable separatorsor borders. Such multichannel testing regions can comprise 1 to 10testing regions, preferably 3, 4, or 5 testing regions, wherein thetesting regions are arranged in a linear or a radial configuration.Arrays of multichannel testing regions can be combined to cover abroader area of a wound or wound dressing. In further embodiments, theouter layer of the wound dressing comprises one or more windows thatpermit visualization of a signal from the reagent layer, wherein thesignal is a color change.

Such wound dressing or device provides a method of detecting the levelof one or more enzymes in a mammalian wound, comprising contacting themammalian wound with the wound dressing; observing one or more signalsin the reagent layer, wherein the signal is a color change; andcomparing the signal to a reference or control to determine the level ofan enzyme. In another embodiment, the wound dressing can be used todetect the presence of one or more enzymes and/or pH in a mammalianwound, comprising contacting the mammalian wound with the wound dressingand observing one or more signals in the reagent layer, wherein thesignal is a color change. In another embodiment, the wound dressing canbe used to treat an infection in a wound of a mammal or to determinewhen such treatment is necessary, comprising contacting the wound with awound dressing described herein, observing one or more signals in thereagent layer, wherein the signal is a color change and indicates thepresence of an infection, and administering a medical treatment to themammal.

In some embodiments, a device for detecting an infection in a woundcomprises a wound contacting layer, a reaction layer comprising one ormore reagents that can indicate the presence of one or more analytesassociated with an infection, wherein the reagents are affixed to asolid phase and produce a detectable signal in a reporter area, a coveron top of the reaction layer, wherein the cover comprises one or morewindows or clear areas to allow visualization of the detectable signal,such as a color change, and fluid communication between the woundcontacting layer and the reaction layer. Reagents includeenzyme-reactive indicators that interact with one or more enzymesselected from the group consisting of lysozyme, MPO, cathepsin G,elastase, catalase, lipase, esterase, and any combination thereof, atleast one indicator for pH or a change in pH, wherein the indicators maybe printed, sprayed, or deposited on a solid phase or support material,including paper, viscose, regenerated cellulose, glass fiber, or similarmaterials. In further embodiments, the enzyme-reactive indicatorscomprise a moiety capable of producing a visible color or a detectableelectronic change upon interaction of the enzyme-labile orenzyme-reactive region with one or more enzymes, wherein the moiety isselected from the group consisting of a peroxidase substrate, arylamine,an amino phenol, a neutral dye, a charged dye, a nanoparticle, acolloidal gold particle, and an analog thereof. In further embodiments,the device comprises wicking stitching or wicking tufting of anabsorbent material to form fluid communication between the woundcontacting layer and the reaction layer.

A device for detection of infection associated enzymes that is providedas an independent entity and can be placed into any dressing or bandagesystem, comprising a sample inlet in fluid communication with reagentcells, wherein reagent cells comprise indicators for sample deliveryand/or pH change, which can be one and the same, and one or moreindicators for biomarkers of an infection, including lysozyme, MPO,cathepsin G, elastase, catalase, lipase, esterase, and any combinationthereof. The fluid communication comprises at least one indicatorchannel, lane, or arm, such as one to ten indicator channels, or one,two, three, four, five, six, seven, eight, nine, or ten separateindicator channels, wherein the indicators are printed, sprayed, ordeposited in a reaction area or field on a carrier material or solidphase and arranged in a radial configuration to form a disk, and whereinthe reaction areas or fields are separated by impermeable separators orlanes. The carrier material may comprise a non-woven material. In someembodiments, the disk comprises reagents printed, sprayed, or depositedon the top surface of the disk with a trap material and a substratematerial on the bottom surface, wherein the substrate can be digested byone or more enzymes in the sample to release one or more products thatmigrate towards the trap. In further embodiments, one or more productsare colored or produce a color change capable of being visualized on thetop surface of the disk.

In additional embodiments, a diagnostic disk for detecting an infectionin a wound comprises a reaction layer comprising one or more reagentsthat interact with an enzyme indicative of an infection, wherein thereagents are affixed to a solid phase; each reagent is sprayed, printed,or deposited in a reagent area separated by impermeable separators; eachlane comprises a reporter area wherein a color change can be observed;and a cover comprising a window for visualizing the color change in thereported area. The diagnostic disk may further comprise at least onereagent that produces a color change in response to a change in pH.Multiple lanes in the diagnostic disk, wherein each lane contains adifferent indicator/reagent, can be arranged in a linear or radialconfiguration about a cut access, perforation, or wicking material thatallows fluid communication between a sample or wound contact materialand the reagents in the reaction layer. The reagents include indicatorsas described above, namely, reagents that interact with lysozyme, MPO,cathepsin G, elastase, catalase, lipase, or esterase. In someembodiments, the diagnostic disk comprises a solid phase materialselected from the group consisting of paper, viscose, regeneratedcellulose, glass fiber, and similar material. In further embodiments,the disk is attached to a non-woven carrier in a wound dressing, whereinmeans for such attachment include, but are not limited to, a continuousadhesive, ring or annular adhesive, welding with UV printed border, andwelding with a polyethylene component or the non-woven carrier.

In further embodiments, the reagents describes herein may be applied toform a lateral flow or dipstick device for detecting an infection in awound, comprising one or more reagent disks arranged in a linearconfiguration, wherein each reagent disk is impregnated with a reagentthat interacts with an enzyme to produce a color change or a similardetectable signal, wherein one of the disks produces a color changebased on pH, and wherein the disks are affixed to a solid phasecomprising paper, viscose, regenerated cellulose, glass fiber, orsimilar materials. Reagents include enzyme-reactive indicators thatproduce a color signal in the presence of lysozyme, MPO, cathepsin G,elastase, catalase, lipase, or esterase. In one embodiment, each disk isseparated by an impermeable border or lane.

In a further embodiment, a standalone device for detecting an infectionin a wound or a sample comprises a housing, comprising: a samplingcomponent for collecting the sample; a sample preparation chamber influid communication with a reaction chamber, wherein the samplepreparation chamber receives the sample; the reaction chamber comprisingone or more reaction cells containing reagents that interact with one ormore enzymes in the sample to indicate the presence of an infectionand/or pH of the sample; and a window or a clear area for visualizing adetectable signal, wherein the signal is a color change or an electronicoutput. One or more reagents interact with an enzyme selected from thegroup consisting of lysozyme, MPO, cathepsin G, elastase, catalase,lipase, and esterase to produce a detectable signal, wherein the signalis a color change. One or more regents produce a color change inresponse to a change in pH, a basic pH, or an acidic pH. In furtherembodiments, the reagents perform the reactions in a primarily liquidmedium, wherein the reagents may be provided in tablet form for use inthe reaction cells. In some embodiments, the reagents may be printed,sprayed, or deposited in separate reagent fields on a support materialto form a panel of tests, such as a testing strip, for use in thereaction chamber. Support materials include paper, viscose, regeneratedcellulose, and glass fiber. Reagent fields can be arrayed in a linealong a plastic or paper carrier strip, which is capable of absorbingthe sample in the reaction chamber, allowing the sample to interact withthe reagents in the reaction chamber. In some embodiments, the samplingcomponent comprises a swab device, or a hook or needle device adapted toremoving a sampling thread from a wound dressing to sample the woundfluid without disturbing the dressing.

In further embodiments, a kit for detecting an infection in a samplecomprises a sampling component for collecting the sample; a test devicecomprising a housing surrounding a tube to define an opening in thehousing for receiving the sampling component, the housing comprising: adiluent chamber that holds a liquid diluent; a reaction well in liquidcommunication with the tube or the sample, the reaction well holding oneor more reagents that interacts with one or more analytes to produce acolor change or similar detectable signal; a viewing window or reporterarea wherein the color change or similar detectable signal can beobserved; and wherein the liquid diluent flows from the sample componentinto the reaction well to mix the sample with the reagents in thereaction well. The reagents comprise one or more enzyme-reactiveindicators and/or pH indicator, as described above. The sample may beobtained from a wound, a wound dressing, or a surgical site. In someembodiments, the sampling component is a swab device or a hook or needledevice. The reagents can be provided in tablet form, which are dissolvedupon contacting the liquid diluent and the sample. The reagents can alsobe deposited in separate fields on a testing strip to form a panel oftests, which can be applied in the reaction wells.

In another embodiment, the reagents are provided in liquid form for usein the reaction wells. The number of reaction wells is based upon thenumber of analytes to be analyzed, ranging from one to ten, includingindicators that produce a detectable signal in response to pH or thepresence of one of the following enzymes: lysozyme, MPO, cathepsin G,elastase, catalase, lipase, and esterase. The reaction wells can bearranged in various configurations, including a linear or a radialconfiguration.

In another embodiment, a wound dressing is disclosed comprising: a woundcontacting layer; a reagent layer comprising one or more testingregions, wherein the reagent layer is in fluid communication with thewound contacting layer; and an outer layer that overlays the reagentlayer.

In another embodiment, a wound dressing is disclosed wherein each of theone or more testing regions comprises one or more of each of a back-flowtrap, a reagent pad, a filter pad, an indicator trap, and an absorbentarea, and wherein one or more viewing windows are located either abovethe reagent pad or the indicator trap.

In another embodiment, a method of detecting the level of one or moreenzymes in a mammalian wound is disclosed, the method comprising:contacting the mammalian wound with a wound dressing; observing one ormore signals in the reagent layer, wherein the signal is a color change,a fluorescent signal, a luminescent signal, or an electrical change; andcomparing the signal to a reference or a control to determine the levelof an enzyme.

In another embodiment, a method of detecting the presence of one or moreenzymes in a mammalian wound is disclosed, the method comprising:contacting the mammalian wound with a wound dressing; and observing oneor more signals in the reagent layer, wherein the signal is a colorchange, a fluorescent signal, a luminescent signal, or an electricalchange.

In another embodiment, a method of detecting an infection in a mammalianwound is disclosed, the method comprising: contacting the wound with awound dressing; and, observing one or more signals in the reagent layer,wherein the signal is a color change, a fluorescent signal, aluminescent signal, or an electrical change.

In another embodiment, a device for detecting an infection in a wound isdisclosed, comprising: a wound contacting layer; a reaction layercomprising one or more reagents that can indicate the presence of one ormore analytes associated with an infection, wherein the reagents areaffixed to a solid phase and produce a detectable signal in a reporterarea; a cover on top of the reaction layer, wherein the cover comprisesone or more windows or clear areas to allow visualization of thedetectable signal; and, fluid communication between the wound contactinglayer and the reaction layer.

In another embodiment, a wound dressing is disclosed wherein the reagentpad is in fluid communication with the filter pad; the filter pad is influid communication with the indicator trap; and the indicator trap isin fluid communication with the absorbent area.

In another embodiment, a diagnostic disk for detecting an infection in awound is disclosed, comprising: a reaction layer comprising one or morereagents that interact with a target enzyme indicative of an infection,wherein the reagents are affixed to a solid phase; each reagent issprayed, printed, or deposited in a reagent area in a lane separatedfrom adjacent lanes by impermeable separators; each lane comprises areporter area wherein a color, color change, or other detectable signalis observed; and a cover comprising a window for visualizing the signalin the reporter area.

In another embodiment, a lateral flow or dipstick device for detectingan infection in a wound is disclosed, comprising: one or more reagentdisks arranged in a linear configuration, wherein each reagent disk isimpregnated with a reagent that interacts with an enzyme to produce acolor change and/or is pH-sensitive, comprising bromothymol blue, phenolred, bromophenol red, chlorophenol red, thymol blue, bromocresol green,bromocresol purple; nitrazine yellow; or other sulfophthalein dyes, andwherein the disks are affixed to a solid phase.

In another embodiment, a device for detecting an infection in a wound ora sample is disclosed, comprising a housing, wherein the housingcomprises: a sampling component for collecting the sample; a samplepreparation chamber in fluid communication with a reaction chamber,wherein the sample preparation chamber receives the sample; the reactionchamber comprising one or more reaction cells containing reagents thatinteract with one or more enzymes in the sample to indicate the presenceof an infection and/or pH of the sample; and a window or a clear areafor visualizing a detectable signal, wherein the signal is a colorchange.

In another embodiment, a kit for detecting an infection in a sample isdisclosed, comprising: a sampling component for collecting the sample; atest device comprising a housing surrounding a tube to define an openingin the housing for receiving the sampling component, the housingcomprising: a diluent chamber that holds a liquid diluent; a reactionwell in liquid communication with the tube, wherein the reaction wellholds one or more reagents that interact with one or more analytes toproduce a color change or a detectable signal; a viewing window orreporter area wherein the color change or detectable signal can beobserved; and wherein the liquid diluent flows from the sample componentinto the reaction well to mix the sample with the reagents in thereaction well.

It is understood that other embodiments and configurations of thesubject technology will become readily apparent to those skilled in theart from the following detailed description, wherein variousconfigurations of the subject technology are shown and described by wayof example or illustration. As will be realized, the subject technologyis capable of other and different configurations and its several detailsare capable of modification in various other respects, all withoutdeparting from the scope of the subject technology. Accordingly, thefigures and detailed description are to be regarded as illustrative innature and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

To understand the present disclosure, it will now be described by way ofexample, with reference to the accompanying figures in which embodimentsand examples of the disclosures are illustrated and, together with thedescriptions below, serve to explain the principles of the disclosure.

FIG. 1: Examples of engineered three-dimensional fabric structures, suchas corrugations.

FIG. 2: Example of a dressing with AQUACEL showing different layers of adressing and stitching that draws fluid from a wound to the reactionlayer of the dressing.

FIG. 3: Schematic of reaction cells showing different components of areaction cell with stitching (21) in (A) and cut access (27) in (B). Insome embodiments, each reaction cell can be a different reporter or dyesystem.

FIG. 4: Movement of indicators in reaction cells upon exposure to fluid,which flows from cut access and reagents (22) toward absorbent orevaporation area (25). Over time, the reaction products diffuse andmigrate toward an absorbent or evaporation area. Movement of indicatorsarranged in a radial manner is shown in (B). In some embodiments, eachlane or reaction cell can be a different reporter or color system.Multiple reaction cells can be used as shown in (C). Multiple reactioncells can be used in arrays or combinations to provide indicatorfunction over an area. Leach back traps may be used to prevent backflow.

FIG. 5: Indicators can be arranged in a circular or radial manner toform indicator disks (A). In some embodiments, each lane or reactioncell (45-48) can be a different reporter or color system, such asbromothymol blue, phenol red, bromophenol red, chlorophenol red, thymolblue, bromocresol green, bromocresol purple; nitrazine yellow; or othersulfophthalein dyes. (B) shows views of a radial indicator disk fromabove and from below.

FIG. 6: Dressing printed for MPO detection. In one embodiment, a woundcontact material is sprayed or printed with amylase, starch, and glucoseoxidase, followed by printing of a substrate for MPO printed in thecenters of each sprayed area.

FIG. 7: In-place color development of MPO and elastase substrates ontesting strips are shown. These test strips represent prototypes ofvisualization methods for detecting the presence of MPO and elastase ina sample, wherein color (e.g., blue) increases in intensity with greatersubstrate concentration.

FIG. 8: Examples of substrates, including MPO substrate (Fast Bluederivative), elastase substrate, and oxidation of indoxyl to bluecolored indigo are shown.

FIG. 9: In-place color development of different indicators in radialarrangement. (A) and (B) represent prototypes of indicators fordetecting certain analytes, including pH change, MPO, lysozyme, andelastase. In one embodiment, pH change can be reported as a color changefrom yellow to green; MPO reported as an appearance of a blue color;lysozyme reported as an appearance of pink or red color; elastasereported as an appearance of green or blue color; and liquid controlreported as an appearance of a blue or purple color.

FIG. 10: Schematics of a radial indicator insert or disk.

FIG. 11: Schematics of a radial indicator insert or disk with a windowfor detection.

FIG. 12: Schematics of another embodiment of a radial indicator insertor disk with a window for detection.

FIG. 13: Transport of Remazol Brilliant Blue, showing migration ofindicators to reporter area after liquid transport.

FIG. 14: Example of a pH indicator. In one embodiment, the color canchange from green to blue with increase in pH.

FIG. 15: Schematic of a lysozyme test strip. Fluid flow causing stainedpeptidoglycan particles to move upwards to trap layer.

FIG. 16: Examples of indicator substrates and reactions.

FIG. 17: Example of indicator disk freely placed in a dressing.

FIG. 18: Embodiments of diagnostic disks in non-woven layer in dressing.

FIG. 19: Embodiments of diagnostic disks in non-woven layer in dressing.

FIG. 20: Example of manufacturing diagnostic disks in sheets.

FIG. 21: Embodiments of printed and applied paper disks. In someembodiments, each disk can be a different reporter or color system.

FIG. 22: Methods of attaching or applying diagnostic disks to non-wovenlayer in dressing.

FIG. 23: Dipstick devices with indicator inserts or disks arranged indifferent arrays and combinations are shown. In some embodiments, eachinsert, disk, or lane can be a different reporter or color system.

FIG. 24: Sampling thread and use in dressing. Sampling thread can beincorporated in a wound dressing or at a surgical site, wherein thethread can be pulled out without disturbing the dressing to test for thepresence of microbial infection or condition of the surgical site orwound in a diagnostic device.

FIG. 25: Assembly for manufacturing indicator inserts.

FIG. 26: Cross section of a standalone device kit

FIG. 27: Sampling tip inserted in the housing of standalone device kit

FIG. 28: A plan view of the standalone device kit

FIG. 29: Another view of the standalone device kit

FIG. 30: A plan view of the standalone device kit with housing slidapart

FIG. 31: Diluent chamber, tube and reaction chamber in standalone devicekit

FIG. 32: Distribution of test solution to each reaction chamber instandalone device kit

FIG. 33: Diagnostic swab device with housing, wherein reaction withindicator disks or inserts can be observed from a viewing window in thehousing.

FIG. 34: Thread hook diagnostic device, suitable for pulling out asampling thread from a dressing for analysis.

FIG. 35: Swab diagnostic device, wherein a swab is used to obtain asample for testing with a diagnostic device, further comprising adiluent chamber, gas outlet, and a plunger.

FIG. 36: Diluent chamber for sample preparation. A diluent chambercomprising a diluent is adapted for use with a swab device, a threadhook device, and similar sample preparation devices, comprising aresealable top and a seal or film at the bottom, wherein breaking theseal or film (402) allows the sample to mix with the diluent solution,which flows out of the diluent chamber and into a testing devicecomprising reaction chambers or wells.

FIG. 37: Embodiment of diagnostic device with sampling chamber andreaction wells. One embodiment of a diagnostic device with reactionchambers (502) adapted to being connected to sampling chamber or diluentchamber (202) for receiving a sample from a sample preparation device(300), such as the swab device.

FIG. 38: Embodiment of diagnostic device or transfer system, wherein thesample chamber or diluent chamber uses a Luer-lock connector to attachto reaction chambers for testing a sample fluid. In one embodiment, theplunger or piston comprises a gas outlet, hook for holding a sample, andmembrane that lets out gas as the plunger is depressed into the diluentchamber.

FIG. 39: Further embodiments of an analytic or diagnostic system,wherein reaction chambers are arranged in a radial arrangement.

DETAILED DESCRIPTION

Various aspects of the disclosed technology will be described more fullyhereinafter. Such aspects may, however, be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein; rather, these embodiments are provided so that thisdisclosure will be thorough and complete, and will fully convey itsscope to those skilled in the art.

Throughout this disclosure, various patents, patent applications andpublications are referenced. The disclosures of these patents, patentapplications and publications in their entireties are incorporated intothis disclosure by reference in order to more fully describe the stateof the art as known to those skilled therein as of the date of thisdisclosure. This disclosure will govern in the instance that there isany inconsistency between the patents, patent applications andpublications cited and this disclosure.

Provided herein are means to detect infections in wounds. In someembodiments these are wound dressings able to detect infection in one ormore body fluids before such infection is otherwise apparent. In someembodiments, the wound dressing reacts with wound exudate or wound fluidto detect infection in a wound through a visible or otherwise detectablechange in the dressing. In some embodiments, wound exudate or woundfluid is drawn up through the wound dressing to a reagent layer forassessment of possible infection without the need to remove thedressing. In some embodiments, wound exudate or wound fluid reacts withthe reagent layer to give rise to a color or other visible or observablemarker. In some embodiments, the color is easily distinguishable fromthose colors that are common in wounds or body fluids. In someembodiments, the reaction between the wound exudate or wound fluid andthe reagent layer of the wound dressing occurs at ambient temperatureand within a period of time short enough to allow timely response, suchas a decision to make a dressing change after cleaning the wound andexamining the test result and/or to administer antiseptics or local orsystemic antibiotics. In some embodiments, the color or other visible orobservable marker and/or the location of the color or other visible orobservable marker indicates one or more areas of the wound that deservecloser attention and/or antisepsis. In some embodiments, the colorchange function is embedded in parts of the dressing that are onlyvisible on dressing change. In further embodiments, the reagent layerthat gives rise to a color change or other visible or observable markeris a standalone device, disk, or insert, capable of application with anywound dressing, at a surgical or wound site, or by itself as adipstick-type of device. In further embodiments, indicator reagents areapplied in a “swab sample preparation device” or a stand-alone deviceinto which wound fluids are injected. In some embodiments, indicatorreagents are printed directly on support materials, such as the variouslayers within a wound dressing.

In some embodiments disclosed herein, a wound dressing comprises a woundcontacting layer; a reagent layer comprising one or more testing regionsor indicator reagents; and an outer layer that overlays the reagentlayer. The wound dressing may comprise one or more testing regions,which further comprise one or more of a back-flow trap, reagent pad, afilter pad, an indicator trap, and an absorbent area, wherein theviewing window is located either above the reagent pad or the indicatortrap and the reagent pad is in fluid communication with a filter pad;the filter pad is in fluid communication with the indicator trap; andthe indicator trap is in fluid communication with the absorbent area.

In some embodiments, testing regions comprise one or more componentsselected from the group consisting of enzyme-reactive indicators,reagents that are sources of peroxide, enzymes that are able totransform color reactions, pH indicators, protein responsive reagents,and moisture-detecting reagents. Enzyme-reactive indicators may compriseprotein-indicator conjugates.

In some embodiments, protein-indicator conjugates are deposited in or onthe reagent pad. In some embodiments, protein-indicator conjugate hasthe structure of Formula (I): A-B, wherein: A is an anchor region forattachment to the testing region; and B is an enzyme-reactive region. Infurther embodiments, the enzyme-reactive region comprises a peptide oran indicator region. The anchor region may be covalently ornon-covalently attached to the reagent pad.

In further embodiments, the wound dressing comprises one or more linesof wicking stitching or wicking tufting throughout all layers of thewound dressing except the outer layer. One or more testing regionsfurther comprises a leach-back trap in fluid communication with thereagent pad, the one or more lines of wicking stitching or wickingtufting crossing through each of the one or more testing regions only atthe leach-back trap. In further embodiments, the wound dressingcomprises a foam layer between the wound contacting layer and thereagent layer. In some embodiments, the wound dressing further comprisesone or more perforations of the wound contacting layer.

In some embodiments, enzyme-labile or enzyme-reactive regions containedtherein may interact with target enzymes including elastase, lysozyme,cathepsin G, and myeloperoxidase. In further embodiments, theenzyme-labile or enzyme-reactive region comprises a moiety capable ofproducing a visible color or detectable electronic change uponinteraction of the enzyme-labile or enzyme-reactive region with one ormore target enzymes, the moiety being selected from a peroxidasesubstrate, arylamine, an amino phenol, an indoxyl, a neutral dye, acharged dye, a nanoparticle, and a colloidal gold particle, and ananalog thereof. In some embodiments, after the target enzyme has cleavedthe indicator from the substrate it is further reacted by an accessoryenzyme selected from a lipase, esterase, hexosaminidase, peroxidase,oxidase, glycosidase, glucuronidase, glucosidase, and laccase, or acombination of one or more thereof.

Applications of the reactive regions may include a device for detectionof infection associated enzymes, on a solid phase such as paper,viscose, regenerated cellulose, glass fiber, mixtures of same or similarmaterial, or arrayed in a line along a plastic or paper carrier strip.

In some embodiments, reagent or indicator inserts or disks for detectionof infection associated with certain enzymes may be provided as anindependent entity and placed into any dressing system comprising asample inlet, diffusion channels toward different areas containingreagents, an indicator for sample delivery and or an indicator of pHwhich may be one in the same, and one or more indicators for thefollowing markers selected from lysozyme, MPO, cathepsin G, elastase,catalase, lipase, esterase.

In some embodiments, the enzyme labile region is labile to a proteaseand the polymer binding domains are selected from cellulose bindingdomains or are hydrophobic binding domains.

In some embodiments, the enzyme labile region is labile to cathepsin orelastase.

In some embodiments, the chemical entity is selected from a smallmolecule entity, a modified oligomer, and a modified polymer.

In another aspect, provided herein is a chemical entity for thedetection of infection in a wound, the chemical entity comprising anindicator region comprising a pH-sensitive moiety that presents avisible color change.

In some embodiments, the chemical entity further comprises an anchorregion wherein the anchor region enables binding of the chemical entityto a support material.

In some embodiments, the pH-sensitive moiety that presents a visiblecolor change at alkaline pH. In some embodiments, the pH-sensitivemoiety that presents a visible color change at neutral pH. In someembodiments, the pH-sensitive moiety that presents a visible colorchange at acidic pH.

In some instances, the pH of a wound can influence many factors of woundhealing, such as angiogenesis, protease activity, oxygen release, andbacterial toxicity. Chronic non-healing wounds may have an elevatedalkaline environment. As the wound progresses towards healing, the pH ofthe wound moves to neutral and then becomes acidic. Monitoring of the pHof the wound may provide a method to assess the condition of the wound(e.g., infection or no infection) and aid in determining a wound'sresponse to treatment.

Accordingly, in some aspect of the disclosed technology, the chemicalentity for the detection of infection in a wound comprises an indicatorregion comprising a pH-sensitive moiety that presents a visible colorchange. In some embodiments, the chemical entity further comprises ananchor region wherein the anchor region enables binding of the chemicalentity to a support material. In some embodiments, the pH-sensitivemoiety presents a visible color change at alkaline pH. In someembodiments, the pH-sensitive moiety presents a visible color change atpH=7.2-9.5. In some embodiments, the pH-sensitive moiety presents avisible color change at pH=7.2-9.0. In some embodiments, thepH-sensitive moiety presents a visible color change at pH=7.2-8.5. Insome embodiments, the pH-sensitive moiety presents a visible colorchange at pH=7.2-8.0. In some embodiments, the pH-sensitive moietypresents a visible color change at pH=7.5-8.5. In some embodiments, thepH-sensitive moiety presents a visible color change at pH=7.5-9.0. Insome embodiments, the pH-sensitive moiety presents a visible colorchange at pH=8.0-9.0. In some embodiments, the pH-sensitive moietypresents a visible color change at pH=7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,9.3, 9.4, or 9.5, or increments thereof.

In some embodiments, the pH-sensitive moiety presents a visible colorchange at neutral pH. In some embodiments, the pH-sensitive moietypresents a visible color change at pH =6.9, 7.0, or 7.1, or incrementsthereof.

In some embodiments, the pH-sensitive moiety presents a visible colorchange at acidic pH. In some embodiments, the pH-sensitive moietypresents a visible color change at pH=4.5-6.8. In some embodiments, thepH-sensitive moiety presents a visible color change at pH=4.5-6.5. Insome embodiments, the pH-sensitive moiety presents a visible colorchange at pH=5.0-6.8. In some embodiments, the pH-sensitive moietypresents a visible color change at pH=5.4-6.8. In some embodiments, thepH-sensitive moiety presents a visible color change at pH=5.4-6.5. Insome embodiments, the pH-sensitive moiety presents a visible colorchange at pH=4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, orincrements thereof.

In some embodiments, the pH-sensitive moiety is bromothymol blue, phenolred, bromophenol red, chlorophenol red, thymol blue, bromocresol green,bromocresol purple; nitrazine yellow; or other sulfophthalein dyes.

Other embodiments include reagents printed on dressing or solid supportmaterials, dipstick devices with indicator disks arranged in variousarrays, and devices with separate sample preparation chamber thattransfer a sample of a bodily fluid or wound fluid to a standalonediagnostic device that uses reagent pills, solutions, or disks inreaction chambers for detecting biomarkers associated with microbialdetection. In further embodiments, indicator reagents are printed,sprayed, or overlayed on support materials, such as dressing, wounddressing, bandage, filter paper, and test strips.

Generally, when a pathogen encounters the human body interior, cellsreact through innate receptor systems, either to injury, toxins, or tothe bacterial cell wall. All of these recognition events result in therecruitment of innate immune cells. These cells are stimulated bypathogens like bacteria to activate bacterial killing systems that arenormally present in polymorphonuclear leukoctyes (PMNs) and are mainlyenzymatic in character. The cells engulf bacteria and lyse them withenzymes that hydrolyze proteins (e.g., protease, elastase, cathepsin G)and cell walls (lysozyme), or mediate protein denaturation (NADPHoxidase, xanthine oxidase, myeloperoxidase (MPO)). These PMNs aregenerally only short lived and will themselves lyse in the area of theinfection. When they lyse, they release the contents of their lysosomesincluding the enzymes.

These enzymes are, therefore, biomarkers for the presence of myeloidcells, and PMNs in particular. A rising level of these enzymes in thewound fluid, therefore, corresponds to a heightened bacterial challengeand one that is not being adequately met by the innate defense. Theassociation of these enzyme levels with clinical infection has beenvalidated using a clinical trial approach (Blokhuis-Arkes et al., 2015).

In addition, the pH of a wound can influence many factors of woundhealing, such as angiogenesis, protease activity, oxygen release, andbacterial toxicity. Chronic non-healing wounds, and those that areinfected or at risk of infection, typically have an elevated alkalineenvironment. As the wound progresses towards healing, the pH of thewound moves to neutral and then becomes acidic. Monitoring of the pH ofthe wound may provide a method to assess the condition of the wound(e.g., infection or no infection) and aid in determining a wound'sresponse to treatment.

A typical lateral flow device utilizes the concept of lateral liquidflow in order to transport a given sample to the test. The benefits oflateral flow tests include rapid results, long-term stability and lowcost to manufacture. These features make lateral flow tests well-suitedfor applications involving drug testing in urine, in particular withrapid point of care testing in hospitals and doctor's offices being anadvantage. A test strip can be dipped directly in the sample which istaken in a liquid form. The sample travels up the lateral flow strip andbinds to available antibodies, which causes a reaction that can bevisually detected on the strip. Applying this technology to samplesother than urine or blood has however been problematic.

Early detection of markers for infection in wounds has advantages inthat treatment of infection can be commenced before the infectionbecomes established and other signs of infection become apparent, forexample, discharge from the wound, redness, pain and unpleasant odor. Adifficulty in testing for markers in wound fluid is that wound fluiddiffers greatly in its consistency and quantity. For instance it can bescant but viscous making the use of a lateral flow test difficult.

Thus it would be desirable to have a single kit for collecting andtesting a sample of fluid taken from a wound that is easy to operate andnot limited by the type or quantity of exudate from the wound. Oneembodiment of the standalone device kit described herein mitigates theabove problems in a kit which comprises a sampling component and a testdevice where the test device does not rely on a lateral flow strip tomove the sample through the device and achieve a diagnosis.

Wound Dressing

In some embodiments, the wound dressing comprises a wound contactinglayer; a reagent layer comprising one or more testing regions; and anouter layer that overlays the reagent layer. In some embodiments, thewound dressing further comprises a protective cushioning layer (forexample a foam or a nonwoven layer) between the wound contacting layerand the reagent layer. In some embodiments, the wound dressing furthercomprises one or more lines of wicking stitching or wicking tuftingthroughout all layers of the wound dressing except the outer layer. Insome embodiments, the wound dressing comprises perforation through thewound contacting layer, the protective cushioning layer, or acombination of both. In some embodiments, such perforation allows forwound fluid transfer from the wound to the reagent layer.

Wound Contacting Layer

When in use, the wound contacting layer of the wound dressing absorbswound exudate and/or wound fluid. In some embodiments, the woundcontacting layer comprises gel-forming polymers or hydrofiber.Gel-forming polymers include, but are not limited to cellulose,carboxymethylcellulose (CMC), carboxyethylcellulose, oxidized cellulose(or a derivative thereof), cellulose ethyl sulfonate, other chemicallymodified cellulose, pectin, alginate, chitosan, modified chitosan,hyaluronic acid, polysaccharide, or gum-derived polymer, or anycombination thereof. In some embodiments, the wound contacting layer maycomprise polyvinylpyrrolidone, polyvinyl alcohols, polyvinyl ethers,polyurethanes, polyacrylates, polyacrylamides, collagen, gelatin ormixtures thereof. In some embodiments, the wound contacting layercomprises fibers of gel-forming polymers. In some embodiments, the woundcontacting layer comprises a nonwoven layer of gel-forming fibers.

In some embodiments, the wound contacting layer further comprisesnon-gel-forming polymers. In some embodiments, the wound contactinglayer comprises cellulose (for example, Lyocell), modified cellulose(for example, viscose or rayon), Polyester, silk, wool, Nylon,Polypropylene, Elastane or mixtures thereof.

In one embodiment, the thickness of the wound contact layer is from 0.1to 10 mm, in a preferred embodiment it is from 0.1 to 5 mm and in astill more preferred embodiment it is from 0.3 to 3.5 mm.

Protective Cushioning Layer

In some embodiments, the protective cushioning layer provides mechanicalprotection of the wound and also assists in the management of excessexudate by acting as a large surface area for evaporation. In someembodiments, the protective cushioning layer may also serve as thematerial that accepts fluid exiting reagent layer or device and may addfunctionality by pulling or directing fluid through the reagent layer ordevice. Suitable materials include foams, (non-gelling) fiber fleeces,(non-gelling) nonwoven fabrics, and engineered three-dimensional fabricstructures, such as corrugations. Examples of engineeredthree-dimensional fabric structures are shown at FIG. 1. Preferably,materials used for the protective cushioning layer possess mechanicalcushioning properties that are unaffected or are minimally affected bycontact with wound exudate. In some embodiments, the protectivecushioning layer comprises plastics based on olefins or olefin derivedpolymers, such as polyethylene, polypropylene, nylon, polyurethane,polystyrene and polyvinyl chloride. In some embodiments, these materialsmay further comprise agents such as surfactants or absorbents thatimprove their wettability.

In some embodiments, hydrophilic polyurethane foam is 2.5 mm (+/−0.5 mm)thick, with a density of 90 kg/m³ to 150 kg/m³, absorption of ≥12 g/g.

Wicking Stitching and/or Wicking Tufting

In some embodiments, the transfer of wound fluid to the reagent layer isoptimized by fiber tufts from the wound contact layer to the reagentlayer. In some embodiments, gel forming polymers from the wound contactlayer can be used as the transport mechanism of fluid from the wound toreagent layer. In some embodiments, the increased hydrophilic nature ofgel forming polymers in comparison to materials within alternate layersof the dressing allows enhanced wicking action to the reagent layer.

In some embodiments, yarns can be used to provide capillary action offluid from the wound contact layer to the reagent layer. This can beachieved using stitching of one or more layers of the dressing or usingtufting of yarn through one or more dressing layers.

In some embodiments, the wicking stitching and/or wicking tufting isselected from various fibers that are wettable and exhibit capillaryaction. Such fibers include, but are not limited to, cotton, rayon,viscose, wool, silk, polyester, polyamide, and CMC fibers, solid andhollow fibers. In some embodiments, the wicking stitching comprisescotton, polyester, polyamide, polypropylene, or a combination thereof.In some embodiments, using increased number of plies or multifilamentyarn, increased linear density of yarn, and/or decreased linear densityof fiber may enhance capillary action of yarn. In some embodiments, thewicking stitching comprises cotton. In some embodiments, the wickingstitching comprises polyester. In some embodiments, the wickingstitching comprises polyamide. In some embodiments, the wicking tuftingcomprises CMC fibers. In some embodiments, the wicking occurs across allareas of the dressing layers. In some embodiments, the wicking isconcentrated immediately beneath or adjacent to the reagent layer toprovide focused, enhanced wicking action and/or reaction with thereagent layer.

In some embodiments, stitching of yarn through hydrofiber and/or foamlayer using hydrophilic yarn provides wicking capacity. The wound fluidcan be wicked up by yarns in a more direct route to the printedsubstrate or reaction layer. Increase in yarn linear density may allowmore of a decrease in wicking time and/or amount of fluid required.

In some embodiments, needling of hydrofiber-foam laminate in wounddressing creates tufts of hydrofiber on the foam side of the dressing.Variable parameters of needling include punch density and penetrationdepth, such as 10-100 punches/cm² at 1-10 mm penetration, 20-90punches/cm² at 2-9 mm penetration, 30-80 punches/cm² at 3-8 mmpenetration, 40-80 punches/cm² at 4-8 mm penetration, 50-80 punches/cm²at 5-8 mm penetration, 60-80 punches/cm² at 6-8 mm penetration, 70punches/cm² at 6 mm penetration. Channels of hydrofiber are createdthrough the foam, leading to vertical wicking of fluid. Hydrofiber tuftsmay enable quicker fluid and enzyme transfer. Type of needles used fortufting include felting (crown), felting (regular), and fork. In someembodiments, use of felting needles allowed gelling fiber tufts to becreated through the foam layer without causing a detrimental effect onthe foam or gelling fiber. Penetration depth may be 1 mm, 2 mm, 3 mm, 4mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm or 12 mm, or at least 6mm, or less than 7 mm, less than 8 mm, less than 9 mm or less than 10mm. Preferably, penetration depth is 6 mm, which enabled an 18% decreasein vertical wicking time at 70 p/cm² punch density. As punch densityincreases, more hydrofiber tufts are created on the foam layer. Enhancedfluid transfer was seen in all punch densities at 6 mm penetrationdepth.

In some embodiments, stitching of yarn through hydrofiber and/or foamlayer using hydrophilic yarn provides wicking capacity. Stitches may beabout 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm,about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12mm, about 13 mm, about 14 mm, about 15 mm, at least about 5 mm, lessthan about 6 mm, less than about 7 mm, less than about 8 mm, less thanabout 9 mm or less than about 10 mm. The wound fluid can be wicked up byyarns in a more direct route to the printed substrate or reaction layer.Increase in yarn linear density allows more of a decrease in wickingtime and/or amount of fluid required. Short stitches (less than 3.5 mm)do not reduce wicking time/volume required to wick through the foamlayer. Stiches may be 5 mm to allow a reduction in wicking time by about45%. In some embodiments, hydrofiber-foam laminate material withcombined thickness of 4.3 mm was tested for stitching with two types ofyarn: high wicking polyester (continuous filament) and standardpolyester thread. Three stitch lengths were tested, including 2.5 mm,3.5 mm, and 5.0 mm. Incorporating stiches enhances fluid transfer, whileincreasing stitch lengths reduced vertical wicking time.

Perforation

In some embodiments, the wicking action of the various layers of thedressing, such as the gel-forming wound contacting layer and the foam,is adequate as it is with the factory porosity and no further treatment.In other embodiments, the wicking action can be enhanced by fineneedling to create channels that have capillary action. In someembodiments, the needling can occur across all areas of the dressinglayers to provide generally enhanced capillary action. In someembodiments, the needling is concentrated immediately beneath oradjacent to the common entrance to the reagent layer to provide focused,enhanced capillary action. In some embodiments, the perforation occursthrough all layers of the dressing. In further embodiments, theperforation occurs in the one or more layers between the wound contactlayer and the reagent layer. In some embodiments, capillary action canbe enhanced by increasing the punch density of the needling to producehigher number of perforations per unit area.

Perforations allow direct fluid transfer through hydrofiber and/or foamlayers to the printed substrate layer. The larger the hole, the morefluid may be transferred, reducing the wicking time/volume required forthe fluid to interact with the printed substrate layer. However, if thehole is too large, fluid handling capacity of the dressing may beaffected. Gelling fibers swell upon hydration and may obstruct theperforation channel of the gelling fabric. Perforations may be formedusing a hypodermic needle. At a higher density, the vertical wickingtime can be reduced by about 28%. In some embodiments, the verticalwicking time is reduced by about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45% or about 50%.

Reactive or Reagent Layer

In some embodiments, the wound contact layer, or the layer supporting itcontains a material that reacts to wound exudates to indicate potentialinfection, or a reactive layer. A reactive layer may comprise one ormore dyes and/or the reagents necessary to support these reactions. Inone embodiment, these dyes comprise amino acids, peptides, or proteinsconjugated to dyes with strong ionic functions, strong contrastingcolors, or the ability to form colors, such as indoxyl/indigo. In apreferred embodiment, addressing includes a layer within the dressingprinted with an immobile trapping material to which said dyes bind. Thislayer is optionally in the outer part of the dressing or at variouslevels within the dressing such that it may be observed without dressingchange, or at dressing change.

In another preferred embodiment, the reactive layer is comprised of anMPO substrate, glucose oxidase and an energy source, such as glucose orstarch, and gamma-amylase. In another embodiment, the dressing containsparticles comprised of chitosan or a derivative that releases dyes onhydrolysis by lysozyme. These dyes may be highly charged or otherwisefunctional to allow their accumulation at sites of signalinterpretation. In yet other embodiments, the reactive layer comprisescompounds such as p-aminophenol, ABTS (2,2inophenol, ABTS (strate. Insome embodiments, acid) diammonium salt), 3,3′-diaminobenzidine, 3,4diaminobenzoic acid, DCPIP, N,N-dimethyl-p-phenylenediamine,o-dianisidine, p-phenylenediamine, 4-chloro-1-naphthol,o-phenylenediamine N-(4-aminobutyl)-N-ethylisoluminol,3-amino-9-ethylcarbazole, 4-aminophthalhydrazide, 5-aminosalicylic acid,2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), indoxyl, indigo,Fast Blue RR, 4-chloro-7-nitrobenzofurazan. In some embodiments, thereactive layer comprises an arylamine. In some embodiments, the reactivelayer comprises an amino phenol. In some embodiments, the reactive layercomprises an amino phenol an aminophenol ether. In some embodiments, thereactive layer comprises an indoxyl. In some embodiments, the reactivelayer comprises an a neutral dye. In some embodiments, the reactivelayer comprises a charged dye, e.g., a dye selected from remazolebrilliant blue, toluidine blue, reactive black 5, remazol brilliantblue, reactive violet 5, and reactive orange 16, or a hydrolytic orammonolytic derivatives thereof, toluidine blue, reactive black 5, orahydrolytic or ammonolytic derivatives thereof; reactive violet 5, orhydrolytic or ammonolytic derivatives thereof; reactive orange 16, orhydrolytic or ammonolytic derivatives thereof; a dichlorotriazine-basedreactive dye such as reactive blue 4, reactive red 120, reactive blue 2,reactive green 19 and reactive brown 10. In some embodiments, thedichlorotriazine-based reactive dye appears black.

In particular embodiments, the reactive layer comprises compounds suchas a reactive dye containing asulfonylethyl-hydrogensulphate-reactive-group. In some embodiments, thereactive dye is reactive black 5, remazol brilliant blue, reactiveviolet 5 or reactive orange 16. In some embodiments, the reactive dye isreactive black 5. In some embodiments, the reactive dye is remazolbrilliant blue. In some embodiments, the reactive dye is reactive violet5. In some embodiments, the reactive dye is reactive orange 16. In someembodiments, the reactive dye is reactive black 5, remazol brilliantblue, or reactive violet 5. In some embodiments, the reactive dye isreactive black 5 or remazol brilliant blue.

In some embodiments, the reactive layer comprises a nanoparticle. Insome embodiments, the reactive layer comprises a colloidal goldparticle. In some embodiments, the reactive layer comprises a chargeddye, an indole derivative, or a luminol derivative. Especially, thereactive layer comprises a dye containing asulfonylethyl-hydrogensulphate-reactive-group, e.g., reactive black 5,remazol brilliant blue, reactive violet 5 or reactive orange 16, or acombination thereof, or a dye containing a dichlortriazinereactive-group, e.g., reactive blue 4, reactive red 120, reactive blue2, reactive green 19 and reactive brown 10, or a combination thereof.

FIG. 3 shows two embodiments of a reaction cell, comprising indicatorunits or testing regions. In (A) of FIG. 3, stitching (21) using wickingfibers helps to draw wound or bodily fluid from a wound toward a reagentpad (22), then through testing regions (23 and 24) and toward absorbentor evaporation area (25). In (B) of FIG. 3, a perforation or cut access(27) is made, such as in the reagent pad (22) to allow the flow of woundfluid from the wound to the reagent pad via capillary action. Thereagent pad (22) may comprise reagents that react with microbialbiomarkers in the wound fluid, such as substrates that react with MPO(29), elastase (30), and lysozyme (31) in the wound fluid. In someembodiments, one or more testing regions may comprise a sulfonic acidfilter pad (23) and a quaternary amine trap (24). In some embodiments,one or more testing regions comprise a leach-back trap (28) and an amineback flow trap or filter (29). Some embodiments contain pH indicators(32) and protein indicators (33) that allow a user to detect a visiblesignal resulting from reactions between microbial biomarkers in thewound fluid and the reagents in the reagent pad (22). Absorbent orevaporation area (25) helps to draw the flow of the fluid from thereagent pad (22) toward (25). In a preferred embodiment, impermeableseparators (26) keep adjacent testing regions separate.

In some embodiments, the indicator trap catches reaction productsbetween the wound fluid and the one or more components selected from thegroup consisting of enzyme-reactive indicators, reagents that aresources of peroxide, enzymes that are able to transform color reactions,pH indicators, and moisture-detecting reagents. In some embodiments, theindicator trap comprises a positively charged or negatively charged trapfor reaction products. In some embodiments, the positively charged trapcomprises a quaternary amine polymer, a mixture of secondary andtertiary amines, other amine-containing polymers, or a combinationthereof. In some embodiments, the positively charged trap comprisespolyDADMAC, or an analog thereof. In some embodiments, the negativelycharged trap comprises polymers or reagents containing carboxy, sulfate,sulfonate, or other acidic chemical groups. In some embodiments, thenegatively charged trap comprises styrene sulfonate. In someembodiments, the indicator trap comprises a total protein indicatorwhich is eluted by wound fluid to indicate overall flow and capacity ofthe testing region. In some embodiments, the control region contains asubstrate for a ubiquitous enzyme such as esterase or carbonicanhydrase, or an indicator for a ubiquitous metabolite like lactate,glucose, ammonia or lipid. In some embodiments, one or more testingregions comprise a sulfonic acid filter pad and a quaternary amine trap.In some embodiments, one or more testing regions comprise a leach-backtrap, a sulfonic acid filter pad and a quaternary amine trap. In someembodiments, each of the one or testing regions is used to evaluate thepresence of one or more analytes and one or more positive or negativecontrol indicators. In further embodiments, the one or more analytes isassociated with enzyme activity. In some embodiments, the enzyme isselected from one or more of the group consisting of elastase, lysozyme,cathepsin G, myeloperoxidase, and leukocyte peroxidase. In someembodiments, the enzyme is elastase. In some embodiments, the enzyme islysozyme. In some embodiments, the enzyme is cathepsin G. In someembodiments, the enzyme is myeloperoxidase. In some embodiments, theenzyme is leukocyte peroxidase.

In some embodiments, the wound dressing comprises a reagent layercomprising one or more testing regions. In some embodiments, the reagentlayer comprises a support material. In some embodiments, the supportmaterial comprises a woven or non-woven material that is capable ofbeing wet by a wound fluid and which displays capillary action. In apreferred embodiment, the capillary action is uniform in the plane ofthe material. In a preferred embodiment, the test regions are arrangedin a circle so that diffusion occurs radially when a liquid is applied.Support material includes, but is not limited to, paper, cellulose,cellulose derivatives, viscose, polyamide, polyester, polyacrylate, andother similar polymers that are useful as fibers, and any combinationthereof. In some embodiments, the support material is cellulose-based,such as refined papers, or non-woven material containing bondedcellulose fibers. In some embodiments, the support material ispolyamide. In some embodiments, the support material is polyester. Insome embodiments, the support material is polyacrylate. In someembodiments, the role of the solid support is to adhere substrates andprovide a field in which analyte enzymes can travel to and interact withthe detector. In some embodiments, cellulose content aids adherence ofthe enzyme substrates, and a significant cellulose or cellulose likecontent is preferred.

In some embodiments, each of the one or more testing regions is printedon or in the support material. In some embodiments, each of the one ormore testing regions comprises an inlet for wound fluid, an area for thewound fluid to react with reagents (e.g., a reagent pad), an area toobserve each product of one or more reactions, and an area for theaccumulation of excess wound fluid (e.g., an absorbent area), which isthen evaporated from an area sufficiently large as to not block due toaccumulated solutes. In some embodiments, the evaporation zone helps todrive pull-through of more wound fluid.

FIG. 4 shows multiple embodiments of the movement of indicators invarious reaction cells. When testing regions in the embodiment of (A) ofFIG. 4 are exposed to wound fluid, wound fluid flows from the reagentpad (22) to absorbent or evaporation area (25), as shown in the rightpanel of FIG. 4(A). The embodiment of (B) of FIG. 4 shows an embodimentof reaction cells wherein indicators are arranged in a radialarrangement, and wherein fluid flows outward from the center uponencountering the reagent pad. The embodiments of (C) of FIG. 4illustrates how multiple reaction cells can be used to cover a broaderarea, with trap leach-back (41) preventing backflow. In someembodiments, each reagent cell or lane of reagent pad (22) may be adifferent reporter or color system, such as bromothymol blue, phenolred, bromophenol red, chlorophenol red, thymol blue, bromocresol green,bromocresol purple; nitrazine yellow; or other sulfophthalein dyes. Inthe presence of wound fluid, in one embodiment reagents interact withanalytes in the wound fluid and migrate or diffuse toward the absorbentor evaporation area (25).

In some embodiments, reagents are used that require trapping of thereaction product, and, to this end, each of the one or more testingregions comprises a reagent pad or a reagent cell (22), a filter pad(23), an indicator trap (24), and an absorbent/evaporation area (25). Inembodiments comprising a color change reagent, each of the one or moretesting regions comprises a reagent pad that is also under a viewingwindow and an absorbent/evaporation area. In some further embodiments,each of the one or more testing regions comprises a leach-back trapwhich is a trap field that contains an absorbent that absorbs thereagents and prevents their back flow to the dressing below. In someembodiments, an outer layer overlays the reagent layer in order tomodulate evaporation of wound fluid, the outer layer containing one ormore windows to visualize the underlying indicator trap and/or reagentpad from one or more testing regions.

In some embodiments, each of the one or more testing regions detects atleast one biomarker. In some embodiments, each of the one or moretesting regions comprises one or more impermeable separators, whereineach of the one or more testing regions detects more than one biomarker.In some embodiments, the one or more impermeable separators are printedstrips of hydrophobic non-permeable material. In some embodiments, theone or more impermeable separators are arranged in parallel lanes. Insome embodiments, the one or more impermeable separators are arranged ina radial pattern. In some embodiments, each of the one or more testingregions detects two biomarkers. In some embodiments, each of the one ormore testing regions detects three biomarkers. In some embodiments, eachof the one or more testing regions detects four biomarkers. In someembodiments, each of the one or more testing regions detects fivebiomarkers. In some embodiments, each of the one or more testing regionsdetects six biomarkers. In some embodiments, each of the one or moretesting regions detects seven biomarkers. In some embodiments, each ofthe one or more testing regions detects eight biomarkers. In someembodiments, each of the one or more testing regions detects ninebiomarkers. In some embodiments, each of the one or more testing regionsdetects ten biomarkers. In some embodiment, each of the one or moretesting regions detects one or more biomarkers.

FIG. 5 shows a radial arrangement of indicators or a radial indicatorpatch. As shown in (A) of FIG. 5, testing regions or reagents may bearranged in a circular or radial orientation. The indicator includesreagents (22), a quaternary amine trap (24), and an absorbent orevaporation area (25). A hole or cut access (27) in the middle of theindicator helps to draw fluid from a wound into the indicator. The fluidtypically will flow from the access (27) outward to the evaporation area(25). When reagents (22) are exposed to wound fluid and react tomicrobial biomarkers, the resulting products migrate to amine trap (24),allowing detection by a user. The indicator may also have impermeableseparators or lanes (26). As shown in (B) of FIG. 5, a top or “above”view is provided and a bottom or “below” view is provided for a radialindicator patch. In one embodiment, substrates may be printed as dots toallow for greater freedom of printing. Moisture impermeable film withadhesive on both sides allows the radial indicator patch to attach tofoam or other support material. In some embodiments, each reaction cellor lane (45-48) can be a different reporter or color system, allowinganalysis of multiple analytes on one indicator patch.

In some embodiments, each of the one or more testing regions comprisesone or more components selected from the group consisting ofenzyme-reactive indicators, reagents that are sources of peroxide,enzymes that are able to transform color reactions, pH indicators, totalprotein-detecting reagents, and moisture-detecting reagents. In someembodiments, the reagents that are sources of peroxide are selected fromperoxy acids, sodium percarbonate, and peroxide-generating oxidases,such as glucose oxidase or lactate oxidase. In some embodiments, theenzymes that are able to assist the transformation of color reactionsare selected from peroxidases and laccases. In some embodiments, one ormore components are immobilized within the one or more testing regions.In some embodiments, one or more components are mobilized by wound fluidwithin the one or more testing regions. In some embodiments, one or morecomponents bind to the one or more testing regions due to interactionwith wound fluid. In further embodiments, each of the one or moretesting regions further comprises one or more of the group consisting ofbuffers, binders, and solubility enhancers. In some embodiments, one ormore buffers, binders, and/or solubility enhancers improves printing orstability.

In some embodiments, each of the one or more testing regions comprisesan enzyme-reactive indicator, further comprising an enzyme-labile orenzyme-reactive moiety, an immobilizing moiety that holds the reactiveindicator in place, and a moiety that gives rise to a visible changeupon interaction of the reactive indicator with a target enzyme. In someembodiments, each moiety is distinctly different from the other. In someembodiments, one moiety incorporates another moiety either partially orentirely. In some embodiments, the reagent pad comprises one or moreenzyme-reactive indicators.

In some embodiments, the enzyme-reactive indicator is aprotein-indicator conjugate such as a protease substrate comprising bothprotein and dye materials. In a preferred embodiment, theprotein-indicator conjugate is a protein with a binding function to asolid phase, such as a cellulose binding domain conjugated with aprotease recognition site and dyes that are released upon proteolysis.

In some embodiments, the pH indicator presents a visible color change atalkaline pH. In some embodiments, the pH indicator presents a visiblecolor change at pH=7.2-9.5. In some embodiments, the pH indicatorpresents a visible color change at pH=7.2-9.0. In some embodiments, thepH indicator presents a visible color change at pH=7.2-8.5. In someembodiments, the pH indicator presents a visible color change atpH=7.2-8.0. In some embodiments, the pH indicator presents a visiblecolor change at pH=7.5-8.5. In some embodiments, the pH indicatorpresents a visible color change at pH=7.5-9.0. In some embodiments, thepH indicator presents a visible color change at pH=8.0-9.0. In someembodiments, the pH indicator presents a visible color change at pH=7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5, or increments thereof.

In some embodiments, the pH indicator presents a visible color change atneutral pH. In some embodiments, the pH indicator presents a visiblecolor change at pH=6.9, 7.0, or 7.1, or increments thereof.

In some embodiments, the pH indicator presents a visible color change atacidic pH. In some embodiments, the pH indicator presents a visiblecolor change at pH=4.5-6.8. In some embodiments, the pH indicatorpresents a visible color change at pH=4.5-6.5. In some embodiments, thepH indicator presents a visible color change at pH=5.0-6.8. In someembodiments, the pH indicator presents a visible color change atpH=5.4-6.8. In some embodiments, the pH indicator presents a visiblecolor change at pH=5.4-6.5. In some embodiments, the pH indicatorpresents a visible color change at pH=4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, or 6.9, or increments thereof.

In some embodiments, the pH indicator is nitrazine yellow, bromocresolpurple or bromothymol blue or an analog thereof.

In some embodiments, the filter pad removes unwanted components of woundfluid, such as fibrinogen, albumins or globulins, and cellularcomponents or non-cellular debris, i.e., dressing components,medicaments, metabolites, microbes, microbial debris, microbialmetabolites, etc. In some embodiments, the leach-back trap preventsbackflow of reagents in the reagent pad or reagent cell from enteringthe inlet for wound fluid in the testing region. In some embodiments,the filter pad and/or leach-back trap comprises a quaternary aminepolymer, a mixture of secondary and tertiary amines, otheramine-containing polymers, or a combination thereof. In someembodiments, the filter pad and/or leach-back trap comprises aquaternary amine polymer. In some embodiments, the filter pad and/orleach-back trap comprises a mixture of secondary and tertiary amines. Insome embodiments, the quaternary amine polymer ispolydiallyldimethylammonium chloride (polyDADMAC or polyDDA). In someembodiments, the mixture of secondary and tertiary amines ispolyethylenimine (PEI). In some embodiments, the filter pad and/orleach-back trap is held in place by cross-linking with bifunctionalreagents, such as epichlorhydrin, diglycidylethers, di-epoxides orarylazideisothiocyanates. In some embodiments, such reagents when mixedwith a reactive amine-containing polymer link different polymer chainsand trap the longer polyDADMAC chains within a matrix. In someembodiments, the trap is composed of choline acrylate derivativespolymerized in situ using a radical initiator such as benzphenone. Insome embodiments, the filter pad and/or leach-back trap comprisespolymers or reagents containing carboxy, sulfate, sulfonate, or otheracidic chemical groups. In some embodiments, the filter pad and/orleach-back trap comprises styrene sulfonate.

In some embodiments, the indicator trap catches reaction productsbetween the wound fluid and the one or more components selected from thegroup consisting of enzyme-reactive indicators, reagents that aresources of peroxide, enzymes that are able to transform color reactions,pH indicators, and moisture-detecting reagents. In some embodiments, theindicator trap comprises a positively charged or negatively charged trapfor reaction products. In some embodiments, the positively charged trapcomprises a quaternary amine polymer, a mixture of secondary andtertiary amines, other amine-containing polymers, or a combinationthereof. In some embodiments, the positively charged trap comprisespolyDADMAC, or an analog thereof. In some embodiments, the negativelycharged trap comprises polymers or reagents containing carboxy, sulfate,sulfonate, or other acidic chemical groups. In some embodiments, thenegatively charged trap comprises styrene sulfonate. In someembodiments, styrene sulfonate is diluted to 0.02 to 0.8% in water andprinted in this form to the support material. In yet other embodiments,styrene sulfonate is diluted to between about 0.01% to 2.0%, about 0.01%to 1.5%, about 0.01% to 1%, about 0.05% to 1%, about 0.1% to 1% or about0.5% to 1%.

In some embodiments, the indicator trap comprises a total proteinindicator which is eluted by wound fluid to indicate overall flow andcapacity of the testing region. This region is distinct from themoisture indicator. In one embodiment, a blue polysulfonate dye, such asEvans or Trypan blue, is weakly bound to a tertiary amine trap. Onarrival of protein, the dye is displaced and re-trapped as a Proteincomplex on a quaternary amine trap. In another embodiment, CoomassieBlue G250 is weakly bound to a Styrene sulfonate field and is displacedby protein to be re-trapped on a quaternary amine trap. The dyeundergoes a mild color change from the sulfonic acid environment to theamine environment increasing the effect. In another embodiment, thevisualization field is pre-printed with the Ponceau S complex of thequaternary amine trap such that it is red indicating non-function. Theconversion of the trap to the Blue form indicates the progress ofprotein elution.

In an embodiment of an indicator of the arrival of fluid in the system,Brilliant Black or a similar dark tetra sulfonate is printed into areagent pad as a free reagent without any polymer complexing. Beingwater soluble, it is readily mobilized by the wound fluid and migratesto the window where it is avidly trapped by a quaternary amine trap. Thehigh polysulfonation increases the avidity for the amine and resistsfurther elution by proteins. Under conditions of high secretion, theeventual removal of the dye from the trap may also serve to indicateexhaustion of the device or a need to change it.

In some embodiments, one or more testing regions comprise a sulfonicacid filter pad and a quaternary amine trap. In some embodiments, one ormore testing regions comprise a leach-back trap, a sulfonic acid filterpad and a quaternary amine trap.

In some embodiments, each of the one or testing regions is used toevaluate the presence of one or more analytes and one or more positiveor negative control indicators. In some embodiments, the one or moreanalytes is associated with enzyme activity. In some embodiments, theenzyme is selected from one or more of the group consisting of elastase,lysozyme, cathepsin G, myeloperoxidase, and leukocyte peroxidase. Insome embodiments, the enzyme is elastase. In some embodiments, theenzyme is lysozyme. In some embodiments, the enzyme is cathepsin G. Insome embodiments, the enzyme is myeloperoxidase. In some embodiments,the enzyme is leukocyte peroxidase.

In some embodiments, a positive result (e.g., indication of infection)from the one or more testing regions is in the form of a visible change.In some embodiments, the visible change is a color. In some embodiments,the color is selected from dark blue, dark green, and black. It is clearto those skilled in the art that the signal effect of the color changedepends on context and practical consideration of interfering colorsfrom the wound itself. Thus, red is a useful signal to indicate aproblem, or to indicate stop or not ready, but it is readily confusedwith colors associated with wound fluids. Thus, colors that are notlikely to emerge from a wound offer potentially less source of error. Insome embodiments, the visible change is fluorescent, luminescent, ormediated by physical means such as electrical, refraction, gas evolutionor polymer state change. Some fluorescent systems have the drawback thatthey require a source of light and potentially a darkened room orchamber for viewing, however, other fluorescent systems do not have suchdrawbacks. Conventional colors are visible under normal treatmentconditions. Given that a color may be diluted or covered by fluids suchas blood, there remains an embodiment in which a dual indicator is usedin which a fluorescent indicator is mixed with a conventional colorindicator. Thus, if a field is covered by blood, the result may beoptionally interrogated with a black-light to determine whether a signalis present.

Outer Layer

In some embodiments, the outer layer comprises a polymer that is noteasily penetrated by wound fluid. Such polymers include, but are notlimited to, a polyolefin, a polypropylene, a polyethylene, polyurethane,polyamides, ethylene-vinyl alcohol (EVOH), acrylonitrile (PAN),polyvinyl choride (PVC), polyvinylidene chloride (PVDC), polyacrylates(e.g., (1-methyl-1,2-ethandiyl)bis[oxy(methyl-2,1-ethandiyl) diacrylate)or other similar hydrophobic impermeable polymers that, in someembodiments, are laid down as films by printing, spraying or filmblowing. In some embodiments, the outer layer is water vapor permeable.In some embodiments, the outer layer prevents moisture loss in specificareas (e.g., where a visible change indicating infection is observed)and promotes moisture loss in other specific areas (e.g., where excesswound fluid accumulates).

In some embodiments, the reaction layer is protected by two layers: atop layer and a bottom layer. The bottom layer typically has an openingthat allows fluid sample inflow. The top layer generally preventspremature evaporation of the sample and may force it to migrate throughthe device to the evaporation zone. The top layer may also contain oneor more windows that allow the response of the reagents to be seen ordetected.

Devices

In yet other embodiments, the disclosure herein provides a devicecomprising a sampling component and a test device comprising:

(a) a housing surrounding a tube to define an opening in the housing toreceive the sampling component, the housing also having disposed withinit:

(b) a sealed diluent chamber connected to the tube and holding a liquiddiluent for removing the sample from the sampling tip to form a liquidtest sample;

(c) a reaction well in liquid communication with the tube, the reactionwell holding a reagent capable of indicating the presence of the analytewithin the test liquid; and

(d) a forcing mechanism capable of moving the diluent through the devicefrom the chamber, over the sample tip and into the reaction well.

In some embodiments, the device operates by driving the diluent over thesample and into a reaction well, and a test solution is made by the flowof the diluent over the sample. Preferably, it is not necessary to firstmix the sample with the diluent to make a test solution and then movethat solution via a lateral flow strip to the reaction well. The movingof the diluent past the sample and to the reaction well means that thekit can be used with a minimum number of steps, for instance taking thesample, inserting the sampling component into the housing and activatingthe moving or driving mechanism. This procedure minimizes user error andthus minimizes false-negative results and misdiagnoses.

In some embodiments, the diluent is forced through the device in aone-step or multi-step process. For instance, in a one-step process, thediluent is forced through the device which creates a test liquid, whichis forced into the reaction well. In a multi-step process, such as atwo-step process, the diluent could first be forced through the deviceto a mixing chamber where a test liquid is prepared. That liquid couldthen be forced from the mixing chamber to the reaction well in a furtherstep.

In another embodiment, the means of mixing and loading the sample may beachieved in a separate step to its analysis. In one embodiment, a sampleswab is first inserted into a recipient fluid container, and then acoaxial plunger is pushed over the swab to eject diluted sample into theanalysis device. In a preferred embodiment, gas is removed, such as byusing Goretex membranes which are gas and vapor permeable but notpermeable to liquid water. Said membranes can be used to degas both thesample as it is injected and to vent the fluid chambers where the assaytakes place.

In one embodiment, preferably the diluted sample is distributed to eachanalysis chamber equally through microchannels. However, when each exitfrom a chamber contains a Goretex membrane, back pressure ensures thateach chamber is only filled once. In a more preferred embodiment, theloss of liquid sample from the assembly is prevented by an absorbentbetween the last outlet and the exterior of the device.

In still other embodiments, the disclosure herein provides a kit fordetecting an analyte or biological marker or target in a samplecomprising:

(i) a sampling component comprising a sampling tip for collecting thesample and

(ii) a test device comprising: a housing surrounding a tube to define anopening in the housing to receive the sampling component, the housingalso having disposed within it: a sealed diluent chamber connected tothe tube and holding a liquid diluent for removing the sample from thesampling tip to form a test liquid; a reaction well in liquidcommunication with the tube, the reaction well holding a reagent capableof indicating the presence of the analyte within the test liquid; and aforcing mechanism capable of moving the diluent through the device fromthe chamber, over the sample tip and into the reaction well.

The sealed diluent chamber may contain a specified volume of diluent sothat an expected volume of test solution reaches the reaction well orwells. In addition the pathway between the diluent chamber and thereaction well is preferably vented at the reaction well end so thattrapped air does not affect the flow of test solution through the deviceor prevent the test solution from reaching the reaction well or preventthe test liquid from correctly filling the reaction well.

The housing preferably has two parts which are capable of moving withrespect to each other while remaining connected to one another. Theaction of moving the parts may provide the forcing mechanism by whichdiluent is moved through the device. The diluent may be driven throughthe device by compression of the diluent chamber which forces thediluent past the sample tip and to the reaction well or wells emptyingthe compression chamber. The compression of the diluent chamber canoccur when the parts of the housing are moved with respect to oneanother such as by sliding one part past another. Alternatively thediluent can be pulled through the device again for example by movingparts of the housing with respect to one another.

The sampling component preferably comprises a handle and a sampling tip,the handle preferably comprising a seal which engages with the openingin the housing to seal the tube when the sampling component is fullyinserted in the tube. The seal prevents escape of the sample and diluentfrom the device reducing the chance of cross contamination from thewound fluid. Preferably the seal and tube engage to lock the samplingcomponent in the device and prevent removal of the sampling componentonce it has been used. This further reduces the chance ofcross-contamination from the sampling component. The sampling componentpreferably activates release of the diluent from the diluent chamber.

The housing may comprise a locking mechanism which locks the housing inposition once the driving mechanism has been activated and preventsreuse of the device. In this way it is immediately apparent that thedevice has been used and cannot be used again. This minimizes falseresults from, for instance, a device that has been mistakenly activatedin transit or from reuse of a device whose reagents have been spent.

Preferably insertion of the sampling component in the device releasesthe seal on the diluent chamber. Preferably the seal is a ball valve orcan be a film or membrane seal or a duck bill valve or other non-returnvalve known in the art which is activated when the sampling component isinserted in the device. The sampling component preferably bursts,punctures or displaces the seal on the diluent chamber.

Preferably the tube is the same or similar size to the sampling tip ofthe sampling component so that the act of inserting the sampling tipinto the tube causes it to be scraped along the walls of the tube aidingthe dispersion of the sample in the diluent once it is released from thediluent chamber and is flushed through the device. The diluent can beflushed along the whole length of the tube or only part thereof. Thesizing of the sampling tip to match the tube also forces the diluent tobe flushed through the tip when the diluent is driven from the diluentchamber. Preferably the tube is wider at its mouth to aid insertion.

Preferably the diluent chamber is shaped like a bellows to assist in thecompression of the chamber alternatively the chamber can be acombination of a plunger and tube similar to that found in a syringe, orsample preparation device, or can be a filled flexible sachet which iscompressed by hand by the user or a balloon which contracts when theseal is released.

Methods of Use

In one aspect, provided herein are methods to diagnose and indicate needfor treatment of chronic wounds using a wound dressing described herein.

In some embodiments, the methods and devices disclosed herein detectbiological markers or targets from body fluid. In some embodiments, thebody fluid is blood, plasma, serum, cerebrospinal fluid, sputum, urineor wound exudate. In preferred embodiments, the body fluid is woundexudate.

In another aspect, provided herein are methods to diagnose chronicwounds using a wound dressing described herein.

In another aspect, provided herein are methods to indicate need fortreatment of chronic wounds using a wound dressing described herein.

In another aspect, provided herein are methods to indicate need fortreatment of surgical or acute wounds using a wound dressing describedherein.

In another aspect, provided herein are methods of detecting biomarkersof infection in wounds using a wound dressing described herein.

In another aspect, provided herein are methods of detecting the pHand/or the presence of biomarkers of infection in wounds using a wounddressing described herein. In some embodiments, the biomarkers ofinfection are leukocyte enzymes. In some embodiments, alkaline pH in thewound indicates infection in the wound.

In another aspect, provided herein are methods of detecting proteaseactivity in wounds using a wound dressing described herein.

In another aspect, provided herein are methods of monitoring thecondition of a wound or surgical site and its healing process or status.

EXAMPLES Example 1: Wound Dressing

One example of a construction of a wound dressing incorporating thedevice is shown in FIG. 2. The wound contact layer in this example iscarboxymethylcellulose marketed as “AQUACEL”, and the AQUACEL is backedby a polyurethane foam. In the infection-indicating area of the deviceis an impermeable area beneath the reagent layer. Connecting to thisarea is a material such as a polyester thread, methylcellulose fibers,or a similar wicking, hydrophilic, capillary or similar material, orcapillary channels. This fluid connection brings wound exudate or fluidinto contact with the reagent layer, where it may react with andmobilize indicator reagents into visible products that are eithervisible in place or trapped in window visible from the outside of thedressing. This example also demonstrates the use of AQUACEL.

In one embodiment of a wound dressing is shown in cross-section in FIG.2. In that wound dressing the wound contact layer (4) comprisescarboxymethylcellulose, marketed as AQUACEL. In FIG. 2, the woundcontact layer (4) is backed by a polyurethane foam (3). In theinfection-indicating area of the dressing is an impermeable area beneaththe reagent layer (2) and above the polyurethane foam (3). Accordingly,in this embodiment the infection-indicating area is provided between thereagent layer (2) and the polyurethane foam (3). Connecting to theinfection-indicating area is a fluid connection (1) component, such as amaterial such as a polyester thread, methylcellulose fibers, or asimilar wicking, hydrophilic, capillary or similar material, orcapillary channels (1). This fluid connection component (1) brings woundfluid into contact with the reagent layer (2), where it may react withand mobilize indicator reagents into visible products that are eithervisible in place or trapped in window (6) visible from the outside ofthe dressing as shown in the top view of the wound dressing shwon in (C)of FIG. 2. As explained above, views (A) and (B) of FIG. 2 show sideviews of the wound dressing (7). View (B) of FIG. 2 shows the flow ofwound fluid (5) from the wound contacting layer (4) at the bottom upwardvia capillary channels (1), which may be formed by stitching usingwicking fibers. The wound fluid reacts with reagents in reagent layer(2), which may contain windows (6), allowing users to observe a visiblesignal resulting from reactions between wound fluid and reagents in thewound dressing. View (C) of FIG. 2 shows a top view of a wound dressing(7), wherein an opaque film on top of the reagent layer (2) containswindows or clear areas (6) that allow the observation of indicators orchanges associated with reagent interaction with an analyte. In someembodiments, a visible signal may be a color change indicative of amicrobial infection in the wound.

Example 2: A Dressing Material Printed with a Patterned Reactive Ink toReport MPO Activity

A dressing wound contact layer has an upper and lower surface in whichthe lower surface is the wound contact layer. Reagents can be sprayed orprinted on a wound dressing material. One embodiment of such dressing isshown in FIG. 6, wherein (A) depicts a view of the surface of the wounddressing material and illustrates the topside of wound contact material;(B) represents the wound material sprayed with amylase, starch, andglucose oxidase; and (C) represents substrate-printed in the centers ofthe sprayed area.

In alternate embodiments, onto the upper surface are printed multiplelayers, such as three layers, to report MPO activity. In one embodimentthe first layer is the substrate which is printed on the upper surfaceof the wound dressing material, such as at a concentration of 30 mg/mLin ethanol/heptane using a line width of 0.8 mm and a print density of 1μL/cm. Alternatively, the fast blue substrate is printed a grid ofcircles each 3 mm in diameter (FIG. 6). In one embodiment the next layeris a spray application of a solution of gamma-amylase and glucoseoxidase immobilized on hydoxypropyl cellulose. The material may besprayed in a water buffer solution such that approximately 3 μg ofglucose oxidase is deposited per cm2, in parallel, 0.5 μg/cm2 of gammaamylase is applied as the conjugate. Once dried, a starch suspension maybe sprayed at a density of 150 μg per cm2. Once printed, the woundcontact layer is preferably bonded to an upper protecting layer. Thesame printing regime can be printed on the upper side an upperprotecting layer. When exposed to artificial wound fluid containingenzymes, the grid becomes blue colored over time.

Example 3: An Absorbent Material Printed with a Patterned Reactive Inkto Report Elastase Activity

In this example a dressing has an absorbent and protective layer whichhas an upper and lower surface in which the lower surface contacts thewound contact layer. Onto the upper surface a grid pattern is printedwith 1 cm grid spacing. In one embodiment, as shown in FIG. 8, the printis performed with a solution of the AAPV-indoxyl ester 30 mg/mL inheptane/butanol using a line width of 1 mm and a print density of 1.3μL/cm. FIG. 7 illustrates embodiments of in-place color development ofMPO and elastase substrates.

Example 4: A Multi-Biomarker Device Insert

The visualization methods are preferably either a color change of animmobile enzyme substrate, directly printed in the window of thereporter area, or of the appearance of an immobilization of thesubstrate caused by hydrophobic properties of the substance andnon-covalent chemical interactions with the carrier material. The amountof applied substrate and possible impregnation mixtures for colorimprovement were tested in this example as described below

Optimization of the reporter area and color signal: Circles (diameter 5mm) were punched out of carrier material, in this case filter paper.Circles were impregnated with different mixtures of buffers (seespecific reagents: Artificial wound fluid 2% bovine serum albumin inphosphate buffered saline containing potassium chloride, urea pH 7.2).See FIG. 8 for examples of substrates in a water solution followed by adrying step. After drying, varying amounts of substrate, usually in anorganic solution, were pipetted on the test circles.

The reactivity to wound fluid was tested as follows: 10 μL test liquid(buffer or artificial wound fluid 2% albumin) with or without enzymewere pipetted on the dried test disks. Disks were incubated either inopen air or in a closed system. Color development was evaluated visuallyat various times after initiation. All observations were at roomtemperature to simulate the condition expected outside the dressing.

After optimization of the two visualization methods, prototypes wereprepared in lab scale to test the interaction of the different enzymesubstrates/their color development. Prototypes were designed andassembled as described in FIGS. 7 and 9.

FIGS. 7 and 9 show embodiments of in-place color development ofdifferent indicators. FIG. 9 shows a prototype with the reporter areasfor lysozyme, elastase and MPO detection, a pH indicator and the liquidcontrol was constructed. On the left portion in (A) the diagnosticmaterial is shown. On the right portion in (A) a magnification of thereporter area is shown. (B) shows the diagnostic area after liquidapplication (artificial wound fluid 0.5% albumin, 1 U/mL elastase, 10μg/mL MPO, 30000 U/mL lysozyme). The experiment was run over 2 h with aflow rate of 100 μL/min for the first 10 min, followed by 10 μL/min. Theexperiment was repeated in n=10.

Embodiments of diagnostic inserts or disks are shown in FIGS. 10, 11,and 12. FIG. 10(A) shows the top view of a diagnostic insert, comprisinga reporter area (60), reaction area (61), and evaporation area (62).FIG. 10(B) shows the bottom layer, comprising an impermeable layer ofplastic film, either white or transparent, with a diameter of about 40mm. The hole in the middle allows for liquid transport and has adiameter of about 4 mm. The bottom layer is covered with adhesive and inthe same shape underneath for an exact fixation on a dressing. FIG. 10shows embodiments of the reaction material comprising an adhesive layer(C) and a reaction layer (D), wherein each arm has a differentsubstrate/indicator and/or pH system. FIG. 10(E) shows the cover, whichcomprises an impermeable white plastic foil with a diameter of 20 mm.The outer ring may have an insider diameter of 25 mm and an outerdiameter of 31 mm. The top layer may be covered with adhesive underneathfor an exact fixation on the reaction material.

Top view of the assembled completed diagnostic insert. See FIG. 10(A).The reporter area is designed as a window surrounded by an off-whitelayer to achieve a maximum contrast to the color signals. In thisembodiment, there are five radial arms, each of which contains adifferent reporter and color system. In one embodiment, three are forenzymes and two are for controls.

The evaporation area ensures a continuous liquid transport through thediagnostic material, necessary for the enzyme reaction and colordevelopment in the reporter area.

Bottom layer as liquid barrier between the dressing and the diagnosticmaterial. Liquid will preferably pass only through the hole in themiddle of the layer which leads to a directed radial distribution intothe arms of the reaction material (diagnostic material).

Diagnostic material was designed with four or five radial “arms”depending on the favored number of enzyme-substrates and controls to beincluded. The reaction material is fixed on the bottom layer withmedical adhesive. Alternatively, the reaction arms are printed or coatedwith the less permeable bottom layer in place of the adhesive (onematerial can serve both purposes).

In some embodiments, the device insert comprises at least one arm orfewer than ten arms. The number of arms may depend on the number ofanalytes to be determined in a sample and control(s), as applicable. Infurther embodiments, the device insert comprises one, two, three, four,five, six, seven, eight, nine, or ten arms.

The reaction material is prepared with impregnation mixtures andsubstrates in accordance to the optimized conditions described abovebefore assembling the detection material.

As shown in FIG. 10(E), the cover has several functions. Firstly, itpreferably maintains the reaction zone moist by preferably preventingpremature drying. Fluids should pass through the reaction area into thereporter area where there is a transparent window that allows colorchanges to be seen. The second function is preferably to avoid a stop ofliquid flow and to cover the chemistry area so that colored reagents arenot seen before they are transported to the window. The cover is waterimpermeable and includes the windows for signal visualization.

The detection material is preferably fixed with a medical adhesive tothe foam backing layer of a hydrofiber dressing.

Optimization of the first visualization method (accumulation andtrapping) established the following conditions:

Trapping mixture: Volume of 1.5 μL per 10 mm2, thickener Methylcellulose(Methocel A4C) max. 1.25%. Drying at room temperature for at least 1 h.

Transport of Remazol Brilliant Blue (FIG. 13) and visualization in thetrap coated reporter area containing the amino-trap (triplicates), testliquid was artificial wound fluid 2% albumin. This visualization methodwas used for the Lysozyme-substrate (results obtained by QZY); releasedand trapped dye after enzyme cleavage: Remazol Brilliant Black) and theliquid control (dye: Brilliant Black BN).

FIG. 13 shows visualization of dye in reporter area (D) after exposureof reaction area (C) to artificial wound fluid. The direction of thefluid flow was from reaction area (C) to reported area (D), furthercomprising amino trap. The experiment was done in triplicates.

Optimization of the second visualization method (in-place color change)led to clearly colored signals for the MPO-substrate, the elastasesubstrate and a pH Indicator.

MPO-substrate: The MPO substrate in this example is a Fast Bluederivative. The substrate is soluble in 50° C. ethanol. After pipettingof 1.5 μL of a saturated solution at the reporter area followed by adrying step (20 min, room temperature) the substrate cannot be mobilizedby artificial wound fluid 2% albumin. The slightly beige MPO substrateis converted by MPO under development to a deep blue to black color inthe reporter area. As the MPO reaction is H₂O₂ dependent, aglucose/glucose oxidase based H₂O₂ generating system is printed in thereaction area.

Optimized conditions led to the results shown in FIG. 7. Test circlescontain 1.5 MPO substrate as described above, 10 μg glucose and 1 μL of0.1% glucose oxidase (1 μg) in water. After drying of the test circles 5μL test liquid (artificial wound fluid 2% albumin, pH 7, without/withMPO) were applied. The picture of FIG. 7 was taken after 2 minincubation time.

Elastase substrate: The elastase substrate consists of an Fmoc protectedAAPV enzyme recognition motif (amino-acid sequence AAPV) esterified toan Indoxyl moiety. It is soluble in organic solvents, but completelyinsoluble in aqueous solution. After enzyme cleavage, Indoxyl isreleased and immediately oxidized to immobile blue Indigo dye (FIG. 8),visible in the reporter area.

Optimized conditions led to the result shown in FIG. 7. In a first step,the test circles were impregnated with a impregnation mixture (0.25%(w/w) Nonidet, 2% (w/w) decanol in 0.05 M borate buffer pH 8). Thereforthe two-phase solution was mixed until formation of an opalescentdispersion. This dispersion was transferred in a glass container. Thetest circles were washed in the impregnation mixture for 1-2 min.Thereafter the filter papers were placed on a glass plate and dried for1-2 h at 54° C.

In the next step elastase-substrate (10 mg/mL in acetone) was pipettedon the circles 2 times in 2.5 μL steps until a final amount of 50 μg pertest circle (20 mm2) was applied (FIG. 7). After drying at roomtemperature an elastase assay was performed by addition of 10 μL testliquid (artificial wound fluid 2% albumin, pH 7, with/without elastase).Color development was observed and documented after 15 min incubation atroom temperature.

The pH indicator is a preparation of bromothymol blue in chitosan,containing glutaraldehyde. The mixture is pipetted in the reporter area,after drying leading to a dark yellow and immobile indicator system. Thecolor changes from slightly green (pH 7) to a dark green (pH 8) within30 minutes of liquid flow (artificial wound fluid 2% albumin). See FIG.14 for an example of a pH indicator.

Immobilized bromothymol blue derived pH indicator after running withapproximately 300 μL artificial wound fluid 2% albumin with different pHvalues. pH indicator was applied in amounts of 1.5 μL per 10 mm2 inthree pipetting steps of 0.5 μL.

Production and functionality of the reporter area in prototypes. In thereporter areas of the arms of the diagnostic material for Lysozymedetection and the liquid control, 1.5 μL of the trapping mixture wereprinted. In the reporter areas for Elastase and MPO detection as well asfor the pH indicator, the substrates were applied (FIG. 7, 9).

FIG. 9 shows a prototype with the reporter areas for lysozyme, elastaseand MPO detection, a pH indicator and the liquid control. On the leftthe diagnostic material is shown, on the right a magnification of thereporter areas. FIG. 9 (A) shows an example for a prototype with thereporter areas before liquid application.

FIG. 9 displays the diagnostic area after liquid application (negativecontrol, artificial wound fluid 0.5% albumin without enzymes). FIG. 9(C)shows the diagnostic area after liquid application (artificial woundfluid 0.5% albumin, 1 U/mL elastase, 10 μg/mL MPO, 30000 U/mL lysozyme).The experiment was run over 2 h with a flow rate of 100 μL/min for thefirst 10 min, followed by 10 μL/min. The experiment was repeated inn=10.

FIG. 9 shows a prototype with the reporter areas for lysozyme, elastaseand MPO detection, a pH indicator and the liquid control. On the leftthe diagnostic material is shown, on the right a magnification of thereporter areas. Color signals for the liquid flow control are visible,so it is believed that the method of visualization by trapping andaccumulation works. The order of reaction is generally MPO, thenelastase, then lysozyme. Color change of the pH indicator as well as thecolor development of the MPO and elastase substrates is visible in thereporter area. The in-place color change was established for thesereactions and functionality was demonstrated.

The inserts can be made in many forms including radial designs (FIG.10-12), linear designs and single spot approaches. These vary in whichlayers and patterns are formed. It is generally the goal to make theinsert as small and non-occlusive as possible.

One means to reduce occlusiveness is to reduce the area of film layers.In the embodiments shown in FIG. 10-12, the only occlusive layers arethe lanes themselves. In this version, the round bottom layer isreplaced by only the adhesive. The advantage of the round bottom layeris that tended to support a broader area of the dressing being sampledinto the device. The reduced bottom layer has the advantage ofpermitting more vapor transfer.

Example 4: Lysozyme Responsive Testing Strip

In one embodiment of a means to detect lysozyme activity, a strip of awicking substance like filter paper is printed with both dyedpeptidoglycan (FIG. 15(D), a) and a trap material (quaternary aminefixed with cross-linked PEI) (FIG. 15(D), b). Wound fluid is applied tothe base and allowed to wick up the carrier to point C where itevaporates. Lysozyme, if present, degrades the dyed peptidoglycan andtransports anionic fragments to the trap (FIG. 15(D), b) where they forma line.

In FIG. 15, one embodiment of a lysozyme test strip (50) comprises aWhatman filter 1001/85 that is cut into 0.5 cm×4 cm pieces havingfixation areas (51), evaporation area (52), 3% crosslinked, amino trap(53), substrate area (54), and a stitching area (21) for wicking fluidfrom a wound. Side view (B) shows a wound dressing comprising a teststrip (50), base layer (55), and stitching (21). Top view (C) shows thetest strip (50) adhered to wound dressing (56).

Integration of dyed peptidoglycan into a lysozyme responsive testingstrip (FIG. 15). In some embodiments, a testing strip comprises aWhatman filter 1001/85 that is cut into 0.5 cm×4 cm pieces. 2 μl of thequaternary amine trapping solution is pipetted onto the cellulose filter1.5 cm beneath the upper end of the stripe. 2 μl of a substrateformulation containing 4 mg dyed peptidoglycan in 240 μl 0.5% PEG6000solution in H₂O are pipetted 1 cm above the lower end of the stripe. Themodified strip is incubated at 90° C. for 30 minutes. The test strip isthen ready to use. Alternatively other dyed lysozyme substrates (e.g.dyed chitosan derivatives) can be incorporated into the testing system.In some embodiments, the testing strip comprises a substrate spot, aquaternary amino trap, and a cellulose matrix.

In some embodiments, integration of the lysozyme responsive testingstrip into a dressing for the online detection of early stage woundinfections.

Liquid transport system from the bottom side of the dressing to the teststrip is performed via a polypropylene yarn stitched through the layersof the dressing and the first water impermeable adhesive layer. Whilethe stitching helps the process, it is not essential and the sameresults are obtained without stitching, albeit more slowly. The testingstrip is embedded in between of two water impermeable adhesive layers.An evaporating area is included in the upper region of the strip. Thedetection unit releases the coupled dye in region ‘a’ which is thentrapped in area ‘b’ of the testing stripe and gives a clear visiblesignal upon lysozyme activity.

Material selection for the test strip: Different cellulose basedmaterials can be used as solid matrix for the test stripe. Non-wovenscontaining a defined amount of cellulose can alternatively be used.Schematic representation of the Lysozyme test strip. Attachment of thedetection system to the dressing (FIG. 15). Base layer contains liquidtransfer system to the detection unit. Upper view of the combined baselayer and detection unit.

Example 5: Indicator Reactions

FIG. 16 shows examples of indicator reactions include a substrate withat least two domains A and B, or A and C, connected by a cleavage site(X), which is recognized by enzymes in wound fluid, such as elastase (Eor E2). In some embodiments, peptidoglycan anchor (S) is attached to anenzyme substrate, requiring digestion or breakdown of the peptidoglycananchor (S) by lysozyme (E1) before the cleavage site (X) on thesubstrate can be accessed by an enzyme in the wound fluid. Products (P)of the reactions are colored, giving rise to a color change detectableby a user. In example I, upon exposure to elastase (E) in the woundfluid, the substrate is cleaved at cleavage site X, releasing MPOsubstrate (B), which can react with MPO in the wound fluid and oxidizethe substrate (B) to form a colored product (P). In example II, lysozyme(E1) breaks down peptidoglycan anchor (S) to expose cleavage site (X).Upon exposure to elastase (E2) in the wound fluid, elastase cleaves thesubstrate at cleavage site (X) and releases indole (C), which may beconverted to indigo in the present of oxygen, giving rise to a colorchange. In example III, MPO substrate (B) may be used instead of indole(C) to yield a colored product (P).

Example 6: Indicator Disk

FIGS. 10-12 show schematics of indicator inserts or disks. FIG. 10(A)shows the top view of a diagnostic insert, comprising a reporter area(60), reaction area (61), and evaporation area (62). FIG. 10(B) showsthe bottom layer, comprising an impermeable layer of plastic film,preferably either white or transparent, with a diameter of about 40 mm.The hole in the middle allows for liquid transport and has a diameter ofabout 4 mm. The bottom layer is covered with adhesive in the same shapeunderneath for an exact fixation on a dressing. FIG. 10 shows thereaction material comprising an adhesive layer (C) and a reaction layer(D) wherein each arm may be a different substrate and/or pH system andwhere the arms in each layer overlap to allow exact fixation. Indicatordisks can have any number or indicator arms, such as 4 or 5 arms ofindicators arranged radially as in FIG. 10. In some embodiments, theindicator disks comprise 1 to 10 arms, or preferably 4 or 5 arms. FIG.10(E) shows the cover, which preferably comprises an impermeable whiteplastic foil with a diameter of 20 mm. The outer ring may have aninsider diameter of 25 mm and an outer diameter of 31 mm. The top layermay be covered with adhesive underneath for an exact fixation on thereaction material.

In the embodiment shown in FIG. 11, (A) shows the bottom layer,comprising a double sided and hydrophobic film (65) with a diameter of40 mm. A hole cut in the middle has a diameter of about 5-6 mm.Reference (66) shows the hydrophobic lanes on non-woven or paper, eitherfull sheet or cut out, placed on the adhesive film. Reference (67) showstraps printed on non-woven or paper which is adhered to the bottom layerwith a back-flow trap (68). In (B), the reaction layer comprises arms,each may have a different indicator and color system as shown in (70).An evaporation cover (71) may be printed, sprayed, or overlaid film.Reference (72) shows the indicator disk affixed to a dressing, whereinouter dressing has a window (shown as dashed line) for viewing theindicator change.

In another embodiment of the indicator disk, as shown in FIG. 12, bottomlayer (A) preferably comprises a white or transparent impermeableplastic film (73) of diameter 40 mm. A hole in the middle of bottomlayer, comprising a diameter of 4 mm allows for wound fluid transport.The bottom layer may be covered with adhesive in the same shape (73) asthe reaction material (77) underneath for an exact fixation on wounddressing, double-sided adhesive and hydrophobic. The reaction layer (77)is placed on top of adhesive layer (73), at the bottom. Each arm of thereaction layer may be 13 mm or 15 mm in length from the center, andabout 5 mm wide. Cut access in the center of the disk may also comprisea back-flow trap (75) to ensure fluid flows from the center outward toevaporation area in the periphery of the insert. Reference (74) showshydrophobic lanes on non-woven or paper, fill sheet or cut out, placedon adhesive. Reference (76) shows traps printed on non-woven or paperwith back-flow trap (75) in the middle. In some embodiments, reactionmaterial (77) comprises brilliant black print, pH indicator, MPOsubstrate, elastase-peptide-indoxyl, and lysozyme-peptidoglycanindicator, and any combination thereof on arms of the indicator disk.Such substrates may be printed on the reaction material or solid supportmaterial. Evaporation cover may be printed, sprayed, or overlaid as afilm over (78), shown as gray box in (78). The reaction material may becovered by a transparent or translucent film, with a window (79,dash-line box) to allow detection of the reaction.

In some embodiments, a cover as shown in FIG. 10(E), comprises a middlecover of impermeable white plastic film with a diameter of 20 mm, anouter ring with an inside diameter of 25 mm and an outer diameter of 31mm, and a top layer covered with adhesive in the same shape underneathfor an exact fixation on the reaction material.

As shown in FIG. 10(A), one embodiment comprises an impermeable whiteplastic foil with an outer diameter of 31 mm, inner diagnostic circle(60, reporter area) with diameter of 25 mm, and the substrate cover (61)with diameter of 20 mm in embodiments using a substrate cover.Evaporation area (62) is located at the periphery of the indictorinsert. A small evaporation area, such as 2×5 mm may be too small for a7-day run, but is sufficient for a smaller run, such as a one-day run.Visible signal resulting from reactions can be detected in diagnosticarea (60) or window reporter area (FIG. 11 or FIG. 12). Such reporterareas can be surrounded by an off-white layer to achieve maximumcontrast to color signals.

In another embodiment, the diagnostic reaction can be performed on asolid phase in which liquid sample diffuses in the vicinity of dyes thatare absorbed onto the solid phase. Enzymes carried in the sample cantransform the dyes through contact in the pores of the solid phasematerial. The changes are visible as color changes. Due to the lowvolumes in use and the high concentration of dye, the color change canbe a sensitive indicator.

In a preferred embodiment indicator disks are prepared by impregnating afilter paper with the reagents and then punching disks prior to adheringthem to a carrier to form a “stick” with a reactive dye coated on to it.This stick can be brought into contact with the sample and a colorchange observed.

In a more preferred embodiment, more than one indicator disk type isplaced onto the stick carrier such that multiple enzymes or parameterscan be detected in one test. Parameters that may be determined includepH, lysozyme, elastase, Cathepsin G, MPO, catalase and lipases. Such astick should also contain a positive control to indicate adequate samplewetting, and or sample application including, in addition to wetting,also the presence of protein.

In one preferred embodiment the indicator disks are aligned in a line ona thin “stick” and the sample is applied to them in sequence using aswab, gauze, or by pressing the stick into or onto a sample, for examplea used dressing.

In another embodiment, the indicator disks are aligned next to eachother on a broad support and their edges on one side are cut such thatthe stick can be pressed with the cut edge to the sample source (i.e. aused dressing or diluted wound fluid, or the edge of a cleaning swab orgauze) such that liquid is taken up into each of the disks at the frontof the broad stick (“Fork” format).

In another preferred embodiment the indicator disks are placed inside acarrier box such that the sample swab can be inserted into the box andthen sealed inside by closing the box. After closure, the sample swabcan be moved and in the process, contacts each sample disk in turn towet them appropriately such that the resulting reaction can be observedthrough windows appropriately placed above each indicator disk. Such anarrangement can preserve the swab for later microbiological examinationand simplify the handling of materials at or during a dressing change.

Indicator disks are preferably prepared with reagents that are capableof color change. Such reagents may be selected from compounds such asp-aminophenol, ABTS (2,2inophenol, ABTS (strate. In some embodiments,acid) diammonium salt), 3,3′-diaminobenzidine, 3,4 diaminobenzoic acid,DCPIP, N,N-dimethyl-p-phenylenediamine, o-dianisidine,p-phenylenediamine, 4-chloro-1-naphthol, o-phenylenediamineN-(4-aminobutyl)-N-ethylisoluminol, 3-amino-9-ethylcarbazole,4-aminophthalhydrazide, 5-aminosalicylic acid,2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), indoxyl, indigo,Fast Blue RR, 4-chloro-7-nitrobenzofurazan. In some embodiments, thereactive layer comprises an arylamine. In some embodiments, the reactivelayer comprises an amino phenol. In some embodiments, the reactive layercomprises an amino phenol an aminophenol ether. In some embodiments, thereactive layer comprises an indoxyl. In some embodiments, the reactivelayer comprises an a neutral dye. In some embodiments, the reactivelayer comprises a charged dye, e.g., a dye selected from remazolebrilliant blue, toluidine blue, reactive black 5, remazol brilliantblue, reactive violet 5, and reactive orange 16, or a hydrolytic orammonolytic derivatives thereof, toluidine blue, reactive black 5, orahydrolytic or ammonolytic derivatives thereof; reactive violet 5, orhydrolytic or ammonolytic derivatives thereof; reactive orange 16, orhydrolytic or ammonolytic derivatives thereof; a dichlorotriazine-basedreactive dye such as reactive blue 4, reactive red 120, reactive blue 2,reactive green 19 and reactive brown 10. In some embodiments, thedichlorotriazine-based reactive dye appears black. In particularembodiments, the reactive layer comprises compounds such as a reactivedye containing a sulfonylethyl-hydrogensulphate-reactive-group. In someembodiments, the reactive dye is reactive black 5, remazol brilliantblue, reactive violet 5 or reactive orange 16, particularly reactiveblack 5. In some embodiments, the reactive dye is remazol brilliantblue, reactive violet 5, reactive orange 16, reactive black 5, orremazol brilliant blue. Especially, the reactive layer comprises a dyecontaining a sulfonylethyl-hydrogensulphate-reactive-group, e.g.,reactive black 5, remazol brilliant blue, reactive violet 5 or reactiveorange 16, or a combination thereof; or a dye containing adichlortriazine reactive-group, e.g., reactive blue 4, reactive red 120,reactive blue 2, reactive green 19 and reactive brown 10, or acombination thereof.

In other embodiments, indicator disks are preferably prepared withreagents that are capable of physical change, e.g., nanoparticle,colloidal gold particle or a luminol derivative.

In a preferred embodiment, MPO is detected using an analog of Fast Blue,or a di-amino phenol as a color generating agent; Elastase is detectedusing a peptide derived indicator including a napthol phenol, indoxyl ora nitro-phenol; Lysozyme is detected using an oligo saccharideconjugated to a dye or color generator, or an oligosaccharide particlecontaining a charged dye in particular said oligosaccharide may beselected from peptidoglycan or chitosan derivatives. Purely as arepresentative example, lysozyme may be detected by visualizing reactiveblack 5, remazol brilliant blue, reactive violet 5 or reactive orange16, reactive blue 4, reactive red 120, reactive blue 2, reactive green19 and reactive brown 10, or a combination thereof bonded to a substratesuch as chitosan, N-acetyl chitosan; oligo-β-D-1,4-glucosamine;acetyl-D-glucopyranoside; N-acetylglucosamine (GlcNAc); glucosaminedimer (GlcNAc)₂; acetyl-chitosan; chitobiose octaacetate; achitooligomer comprising the structure (GlcNAc)_(n) wherein n=4, 5, or6; a chitooligosaccharide; 2-acetamido-2-deoxy-D-glucopyranoside;2-deoxy-3,4,6-tri-O-acetyl-D-glucopyranoside; or a combination thereof.Protease such as human neutrophil elastase or HNE) may be detected byusing a peptide substrate comprising a core sequenceAlanine-Alanine-Proline-Valine (AAPV) which is conjugated to one or moreof the aforementioned dyes.

In another embodiment, the reagents to detect these analytes are subjectto cleavage to yield a compound that is trapped on an immobile portion.

Example 7. Use of an Indicator Dressing in the Context of a WoundTherapy

A dressing containing an indicator disk as described above is preparedin which the printed disks are inserted between the absorbative outerlayer of the dressing and the outer membrane or film such that thereacted areas are visible. The dressing is applied to a wound, be itchronic or surgical, such that sites of secretion in the wound (deepersites, sutures) are located under or as near as possible to centers ofthe disks. See FIG. 17. Following dressing application, the dressingwill begin to absorb secretions. In one embodiment, the firstobservation of wound status can be made after the “flow control” hasturned blue. This is an indicator of the fact that sufficient liquid hasentered the dressing to saturate the reagent pads. If, at this stage oneor more of the biomarker indicators has already reacted, this would bean indicator of the fact that a degree of inflammation or potentialinfection was present in the wound at dressing change. One biomarkerreacting, with or without an indication of pH above neutral, is likelysufficient to justify detailed wound hygiene steps at the next change.Two biomarkers responding with or without a pH above neutral is likelyan indication that in an ideal situation, the wound would be immediatelyre-dressed and anti-microbial approaches initiated. Three biomarkersresponding, with or without pH would likely be an indicator that in anideal situation the dressing should be immediately changed andanti-microbial hygiene, wound dressings and laboratory microbiologyshould be initiated.

In a broad sense the indicators can respond immediately after dressingchange, after 1-2 days and after 2-5 days. Due to the dynamics of flow,the reagents are intended to respond within 2-6 h of exposure to athreshold of enzyme activity, for example 0.5 U/mL elastase, however,long exposure to low enzyme levels, i.e. 5 days, may also ultimatelyengender a signal. Thus, the user can distinguish a low level ofactivity from an acute sign in that the reporter area very slowlyaccumulates signal, i.e. very faint at 3 or 4 days and only slightlymore developed after 4 or 5 days. This would be indicative of a wounddeserving of close observation and hygiene but not necessarily one inacute infection. Experience with the particular patient would alsoinform the therapist. If the same pattern was apparent over multipledressing changes it would suggest a stable situation but that any changein the degree of reaction should be taken as an indication of apotential change in wound status.

In contrast, a situation in which a strong signal suddenly appears ispotentially indicative of the onset of an acute infection. Given thatthe indicator can change within 1-2 hours once a threshold is crossedsuggests that any sudden developments reflect the current situation ofthe wound.

Where multiple infection indicator disks are placed within the wounddressing, the position of those that react is an indicator of where inthe wound potential problems arise. Thus, the absence of clear signalsafter 5 days would be an indication that no thresholds have been crossedin that period and that current therapy may be adequate. Weak signalsthat develop slowly may indicate that hygiene could be improved.Moderate signals that appear gradually after 5 days may be the firstsigns that an infection is developing and should result in moreelaborate therapy. Strong signals that develop over 5 days would becorrespondingly more emphatic indications that therapy need be improved,for example, by instituting silver dressings. The rapid onset of a clearsignal is, in turn, the indicator of an acute issue that meritsimmediate attention.

As shown in FIG. 4(C), multiple reaction cells can be applied to a wounddressing in some embodiments for detection of microbial infection overan area. Amine back flow trap or filter or leach-back trap (41) may beused to separate testing regions.

Example 8. Dressing Inserts that may be Applied to Any Dressing

In some embodiments, indicator insert may be freely placed at a site oflikely secretion or placed anywhere in a wound dressing or a surgicaldressing.

Diagnostic disks, as described above, can be incorporated into adressing during its manufacture. These inserts may be placed between theouter absorbent and the outer film and equally spaced, and glued inplace during manufacture. However, the fixed spacing may not beappropriate to a particular wound. In this example, the reporter disksare prepared as independent materials that can be put on any absorbentdressing below the outer film. For example, the inserts are prepared asstand-alone disks, cut and sealed in sterile outer envelope. Therapistsusing dressings, see reference (92) in FIG. 17, without reporters maystill insert these reporters (90) into such dressings in so far as theseare modular and require the therapist to assemble the dressing from:wound contact material, absorbent, and outer film or cover. The reporterdisk can fulfill its function in many ways, including so long as it isin fluid contact with the wound fluids (91) and otherwise under anappropriate outer dressing. An adhesive transparent outer disk is onemeans of fixing and holding the reporter disk. Similarly, the diskitself may have an adhesive bottom coat.

In another embodiment of diagnostic inserts, shown in FIG. 18, non-wovenlayer in a dressing carries or contains diagnostic disks (705), whereinthe dressing further comprises a film cover layer (701), non-wovencarrier of indicators (702), polyurethane foam (703), and cellulosecontact layer (704). As demonstrated by the arrow in FIG. 18(A), woundfluid flows upward to diagnostic disks (705) embedded in such dressing.FIG. 18(B) shows a side view of the wound dressing with embeddeddiagnostic disks, wherein quaternary amine coating (shown as dashedline) on foam surface acts as trap for preventing return of diagnosticsubstances and that wound fluid flows upward to diagnostic disks.

In a further embodiment of diagnostic disks in wound dressing, as shownin FIG. 19(A), the side view representation (A) shows an example diskfor detecting MPO, wherein (720) is a paper disk impregnated with theMPO substrate through dipping or spray coating. Reference (721) is thepaper or non-woven material that acts as a carrier. Reference (722)shows an adhesive layer. Reference (723) represents a disk containingglucose oxidase and/or starch and an amylase, such as gamma amylase.FIG. 19(D) shows the wound fluid mobilizes starch into glucose, which inturn is oxidized by glucose oxidase to yield H₂O₂. This is used by MPOin the wound fluid to convert the substrate to the detectable blue form.FIG. 19(B) shows the side view of a disk for detecting lysozyme, whereinparticles of chitosan or peptidoglycan are embedded in the paper disk onits lower side using a water permeable adhesive layer that also servesto adhere the disk to the foam layer below. Enzyme activity dissolvesthe particles and releases dye that is trapped and is detectable in thetop layer. In FIG. 19(B), the paper disk (730) is a trap impregnated toplayer. In the presence of wound fluid, as shown by the upward arrow inFIG. 19(C), the paper/non-woven disk acts as a carrier (721) so that thewound fluid moves to the top layer, via stained peptidoglycan particles(731) in the process. Reference (722) shows an adhesive layer. Reference(732) shows an adhesive ring or thermal weld that secures the disk tothe non-woven carrier layer (721). The dashed line in FIG. 19(C)represents quaternary amine coating on foam surface under the diagnosticstrips that acts as a trap for preventing return of diagnosticsubstances. FIG. 19(E) shows stained peptidoglycan particles slowlybeing dissolved by wound fluid and the dye that is released is thencaptured in the trap material while excess wound fluid flows to thesides, as indicated by the arrows. In FIG. 19(E), the paper disk isimpregnated with trap material in the top layer.

In FIG. 20, the scaling up of the production of the disk constructs isdescribed. In the continuous process, the disks are punched from a sheetcomprised of sealing film, the adhesive, the paper or non-woven carrier,which is protected by the top cover sheet.

FIG. 21 shows different embodiments of paper disks. FIG. 21(A) shows thedifferent layers involved in such embodiments, namely, film cover ontop, a non-woven carrier, a polyurethane foam, and a cellulose contactlayer. FIGS. 21(B) to 21(E) show different variants of such analyticsystem with indicator disks. FIG. 21(B) shows non-woven carrier ofindicators with diagnostic disks attached, including pH indicator onpaper, paper disk printed with starch, amylase, and glucose oxidase, andtrap impregnated paper disks. FIG. 21(C) shows partly printed non-wovenand applied paper disks, including trap printed and UV border or trapborder (910). FIG. 21(D) shows partly printed non-woven and gradient(911) application of indicator disks. The gradient is formed by printingconcentric rings of substrate at different concentration, or with adifferent pH mediator. Fully transformed, different substrateconcentrations lead to different color intensity. Alternatively, usingpolymeric buffers in each ring can modulate the degree of reactionrequiring more activity to yield the same color. Suitable buffersinclude polycarbonates and polysulfonates. The number of concentricrings of color provides an indication of overall activity and thus withreference to a color chart can assist in assessing the degree ofseverity. FIG. 21(E) shows one embodiment of the diagnostic disks withprinted indicators (912) and reagents applied on adhered paper disks. Inthese embodiments, the non-woven functions as a carrier of indicators.

FIG. 22 shows different ways diagnostic disks (800) may be attached to adressing. For example, FIG. 22(A) shows continuous adhesive that allowswound fluid to penetrate through the adhesive. FIG. 22(B) shows ring orannular adhesive that allows wound fluid to penetrate via the hole inthe middle of the adhesive layer. FIG. 22(C) shows welding with UVprinted border. FIG. 22(D) shows welding with polyethylene component ofnon-woven.

Example 9: Dipstick—Traffic Light Format

Certain reagents have adequate affinity for paper or similar solidphases and remain substrates for the biomarker enzymes of interest.Where these substrates exhibit color change, the activity of the enzymescan be observed by simply contacting the fluid containing the markerswith the impregnated paper. Capillarity ensures the distribution of thefluid to the substrate. Each impregnated disk can be separately added toa combined “dipstick” which allows all disks to be used in a test (FIG.23). One format is the linear array of disks, although the layout may beeasily varied.

FIG. 23 shows indicator inserts or disks (820) specific for variousenzymes or microbial biomarkers and controls may be placed in variouscombinations or arrangements to form various dipstick devices. Eachimpregnated disk (820) can be separately added to a combined dipstickthat allows all indicator disks to be used in a test. One format is thelinear array of disks, although the layout may be easily varied.Indicator disks may be separated by lanes or borders (821).

In this example, the following disks are prepared:

1. Fluid control: a 5 mm disk of double sided adhesive is punched, and50 μg of a micronized Fast green powder is placed on the adhesive in thecenter. A paper disk is placed over the adhesive disk concentrically,such that the powdered dye is covered by the paper. The resulting diskis then placed in the first position on the carrier stick via the otherside of the adhesive.

2. pH control. Filter paper is soaked in a mixture containingbromothymol blue, chitosan and glutaraldehyde in ethanol as reportedabove. The filter paper is dipped in the mixture, allowed to drip dry,and is then dried on glass at 54° C. 5 mm disks are then punched and thedisks are attached to the carrier with adhesive.

3. MPO indicator. 5 mm paper disks are impregnated sequentially with 1.5μL of the MPO fast blue substrate as described above for the Dressingindicator. Once dried, one half of the disk is impregnated with 10 μg ofglucose and the other half of the disk is impregnated with 1 μg ofglucose oxidase in buffer (PBS).

4. Elastase indicator. Filter paper was impregnated with a mixture(0.25% (w/w) Nonidet, 2% (w/w) decanol in 0.05 M borate buffer pH 8) anddried for 1-2 h at 54° C. 5 mm paper disks are punched from the buffertreated paper and impregnated sequentially with 2 times 2.5 μL of theAAPV indoxyl substrate (10 μg/μL in acetone) as described above for theDressing indicator.

5. Lysozyme indicator. Filter paper is lightly sprayed (1.5 μL per cm2)with a trap solution containing 3% W/V quaternary amine trap and allowedto dry with the top surface identified. A 5 mm disk of double sidedadhesive is punched, and 40 μg of a Brilliant Black stainedPeptidoglycan is placed on the adhesive in the center and allowed todry. A paper disk is placed over the adhesive disk concentrically, suchthat the PG-dye deposit is covered by the paper. The resulting disk isthen placed in the fifth position on the carrier stick via the otherside of the adhesive. The resulting dipstick can have the sample appliedto it by means of swab, or gauze.

Example 10: Dipstick—“Fork” Format

In one embodiment a dipstick is prepared essentially as for the aboveexample with the exception that the reagent disks are oriented to thebase of a thicker carrying card or stick. The ends of the reagent disksare trimmed at the last stage of production such that they are flushwith the bottom edge of the device. This allows them to be pressed ontoa surface to be sampled. The sample then diffuses into the cut end ofthe disks to react. This format is potentially more convenient forsampling surfaces like used dressings.

Example 11: Dipstick—Box Format

In certain instances, suspected infection, or the risk of contaminationbetween patients through consumables and their disposal demands a moresecure system. In one embodiment, where sampling is done via a swab,retention of the swab for subsequent bacteriological evaluation may bedesirable. Similarly, it may be desirable to retain the result anddisplay it to a colleague after a dressing change. In this context, ameans to retain the result without risk of contamination is desirable.To this end, in one embodiment, a sealable container or enclosure may beused for accommodating a plurality of disks, such as 6 disks, in which awet swab can be placed and then closed such that it can apply the sampleto the paper disks but not contaminate any further objects. One suchdesign is illustrated along with its working principle (FIG. 23). Thekey elements of the design are: the well for wetting the swab; itsclosed sealable form; the sealing rings around the stem of the swab; thepressure fins that push the swab to the disks while also making it aone-way movement; the window to the disks; the space for referencecolors on the case, the possibility to re-open in a microbiology lab.

Example 12: Surgical Site Detection

In another embodiment, the dressing is intended for the treatment ofsurgical wounds and contains distinct linear regions intended to beplaced over the line of sutures. These linear regions containparticularly high concentrations of reporter dye such that even in theearliest phases of infection, the signal will be apparent. In anotherembodiment, the dressing contains a removable components such as athread, or similar absorbent that can be withdrawn and tested withoutremoving the dressing (FIG. 24). Said removable component is placed insuch a way as to be located at or near the edges of the surgical wound.In another embodiment, the surgical site dressing is essentiallytransparent in the linear region both to allow observation of thesutures, and the reporter dye. In a preferred embodiment, thetransparent area is covered by an opaque film that may be easily peeledback to examine the wound. In another embodiment the covering andabsorbent material contains a trapping material such as a polymericcation or anion that is capable of binding and concentrating the dyesthat are released.

For example, in FIG. 24, sampling threads (100) are built in or added todressing for a wound or at a surgical site (92). AQUACEL (4) is used insome embodiments of the dressing (92). Sampling threads absorb woundfluid or fluid at surgical site (D). A thread may be pulled out orextracted (E) from dressing without having to remove or disturb dressingusing an instrument or device (101) such as a tweezer, hook, or threadhook device. The thread can then be dissolved in a buffer for use in adiagnostic device (102) using one or more indicator regents or indicatordisks described herein.

In some embodiments, a wound dressing comprises built in samplingthreads. In some embodiments, the sampling threads absorb wound fluidsand may be removed without disturbing the wound dressing for detectionof analytes in the wound fluid.

In some embodiments, the sample threads may be diluted in buffer todissolve markers for diagnosing the status of the surgical site orwound.

In some embodiments, a thread hook device may be used to remove a threadfrom a wound dressing.

Example 13: Manufacture of Dressing Inserts

The reporter inserts are manufactured by the sequential placement ofvarious materials on a solid carrier. This carrier can be a cellulose,viscose, polyethylene, polyamide or other suitable polymer or mixture ofthese components.

FIG. 25 shows indicator inserts may be manufactured or printed in sheetsor reels. FIG. 25 also shows the order of printing, printing of lanes,order on which reagents are laid down, and placement of reagents forprinting disks in sheets or reels, comprising adhesive or backing filmas in FIG. 25(A), applying a non-woven material as in FIG. 25(B), andprinting reagents and lanes on non-woven material as in FIG. 25(C).Completed or assembled inserts, as show in FIG. 25(D) can be separatedor cut before sticking to a dressing or similar support materials.

In one embodiment the material is prepared in a reel to reel format. Thesolid carrier is first printed with guide lanes that penetrate the filmto full thickness. Next, a bottom film that sits under the polymer anddoes not penetrate it is printed, this includes a hole in the centerthrough which sample fluid enters. Next trap material is printed, athalf density around the entrance site (back-flow trap) and at fulldensity in the trapping sites for the flow control and the lysozymesubstrate. Next the flow control ink is applied to the first position ofthe radial arms of the disk, 10 to 50 μg of Brilliant black in 1%methylcellulose is typical. Next the pH reporter, as described above isprinted in position 2. Next, the MPO area is printed sequentially withsubstrate, glucose and glucose oxidase as noted above. Next the elastasesubstrate is applied in sequential prints to reach the appropriate load.Next the lysozyme substrate is printed to position 5 in the reagentlevel (as distinct from the trap level). Finally a film is printed onthe top of the construct but without penetration of the solid carrier.This film occludes only the radial arms from center to the end of thereporter window.

The resulting reel contains a continuous pattern of evenly spacedreporter fields. These continuous printed fields can be directly rolledinto a dressing sandwich between absorbent and outer film, or, they maybe punch cut and packaged for separate use.

FIG. 20 shows another embodiment of manufacturing paper disks. FIG.20(A) shows a side view of a continuous sheet, comprising a cover filmon top, paper in the middle, and backing film at the bottom.Adhesive/particle matrix (901) may be applied between the coverfilm/backing film and the paper layer (900). FIG. 20(B) shows a top viewof cut sheets prepared for application to non-woven carrier by removalof inter disk material prior to placement on non-woven carrier.

Example 14: Manufacture of Reagents for Liquid Based Devices

In certain embodiments, it is desirable to place reagents in devices insuch a way as that they are stable, but readily soluble for access toinjected enzymes. One approach is to dry reagents on disks of paper andinclude the disks in the devices.

Disks are prepared using either a continuous paper or similar materialor textile which is dipped, sprayed or printed, or using pre-cut disksthat are dipped or mixed in a reagent and subsequently dried. See FIGS.20, 25.

The densities of the reagents per 20 mm2 are:

MPO substrate (alkyl-fast blue) 0.6 μg

Glucose 10 μg, glucose oxidase 1 μg

For elastase, paper is first impregnated with impregnation mixture(0.25% (w/w) Nonidet, 2% (w/w) decanol in 0.05 M borate buffer pH 8).

Thereafter the paper is sprayed with a solution of elastase substratecorresponding to 2.5 μg per mm2.

The paper so printed can be punched to yield disks containing thereagents.

These disks can then be incorporated into the devices.

Example 15: Manufacture of Reagents for Liquid Based Devices

Alternatively, the reagents may be pressed into water soluble “pellets”which are then included in the wells of the devices. The pellets cancontain a range of materials in addition to those used on paper.

A liquid based diagnostic device uses pre-formulated reagents togenerate a colour in response to enzyme activity in a sample. The samplemay contain all or only some of the liquid required. Where the sample isto be diluted, the device preferably contains water or buffer suitableto dilute or render the sample homogeneous. The resulting mixture isdistributed to wells which each contain a different reagent set. Thereagents are a mixture of buffer salts, energy source, substrate andassociated chromophores if not contained in the substrate. Thesereagents are ideally delivered in a discreet entity like a tablet orsimilar that can be placed in the wells. Here we describe thepreparation of tablets for enzymatic assays for elastase, lysozyme, MPOand protein standard as internal standard. The tablets dissolve afteraddition of wound fluid and release assay components to start the enzymereactions that lead to colour changes where positive.

A Perkin Elmer electro-hydraulic tablet press is used to form thetablets as follows:

The pressing time per tablet is approximately 10 sec.

The diameter of the filled part of the pressing tool is 5 mm

Tablets are: 20 mg, 5 mm diameter, 1 mm deep

A vacuum is first applied for about 15 sec.

The applied vacuum is maintained until the removal of the pressingtools.

The pressing pressure is adjusted to 2 t.

TABLE 1 List of tablet reagents for use in liquid-based diagnosticdevices. Amount in 20 mg Component (mg) MPO Tablet Na₂CO₃ 0.38 NaHCO₃0.54 Guajacol (CH₃O)C₆H₄OH = Substrate 1.53 Alternatively diaminophenolSodium percarbonat × 1.5 H₂O₂ (Na₂CO₃•1.5 H₂O₂) 0.02 Maltose Monohydrate(C₁₂H₂₂O₁₁•H₂O) 17.53 Elastase Tablet Sodium Acetate (C₂H₃NaO₂) 1.64Sodium chloride (NaCl) 5.84N-Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide 0.24 (C₂₇H₃₈N₆O₉) =Substrate Maltose Monohydrate (C₁₂H₂₂O₁₁•H₂O) 12.28 Lysozyme TabletPotassium hydrogenphosphate (K₂HPO₄) 7.32 Potassium dihydrogenphosphate(KH₂PO₄) 1.09 Peptidoglycan (von Micrococcus lysodeicticus) as a 0.20film or dyed with reactive black as gross particles = Substrate MaltoseMonohydrate (C₁₂H₂₂O₁₁•H₂O) (Filler) 11.39 Internal Standard TabletCitric acid (HOC(COOH)(CH₂COOH)₂ 8.56 Sodium hydrogenphosphat (Na₂HPO₄)1.54 Bromophenol blue (C₁₉H₁₀Br₄O₅S) 0.06 Maltose Monohydrate(C₁₂H₂₂O₁₁•H₂O) 9.84

Example 16: Standalone Device and Kit for Liquid Based Assay

Stand-alone devices and kit for detecting and measuring wound infectionusing the compositions and device are described herein. These devicesand kits preferably comprise a sampling component for collecting asample and a test device. In some embodiments, the test device comprisesa housing surrounding a tube to define an opening in the housing toreceive the sampling component, the housing having within it a sealeddiluent chamber which is connected to an opposite end of the tube andholding a liquid diluent for removing the sample from the sampling tipto form a test liquid. The tube is in liquid communication with areaction well which holds a reagent capable of indicating the presenceof the analyte. A driving mechanism drives the diluent from the chamberpast the sampling tip, into the tube and finally to the reaction well.

In some embodiments, the kit for detecting an analyte in a samplecomprises: (i) a sampling component comprising a sampling tip forcollecting the sample and (ii) a test device, further comprising: ahousing surrounding a tube to define an opening in the housing toreceive the sampling component, the housing also having disposed withinit: a sealed diluent chamber connected to the tube and holding a liquiddiluent for removing the sample from the sampling tip to form a testliquid; a reaction well in liquid communication with the tube, thereaction well holding a reagent capable of indicating the presence ofthe analyte within the test liquid; and a driving mechanism capable ofdriving the diluent through the device from the chamber, over the sampletip and into the reaction well.

The kit operates by driving the diluent over the sample and into areaction well, a test solution is made by the flow of the diluent overthe sample. It is not necessary to first mix the sample with the diluentto make a test solution and then move that solution via a lateral flowstrip to the reaction well. The driving of the diluent past the sampleand to the reaction well means that the kit can be used with a minimumnumber of steps, for instance taking the sample, inserting the samplingcomponent into the housing and activating the driving mechanism. Thissimple procedure minimizes user error and thus minimizes false negativeresults and misdiagnoses.

The sealed diluent chamber may contain a specified volume of diluent sothat an expected volume of test solution reaches the reaction well orwells. In addition the pathway between the diluent chamber and thereaction well is vented so that trapped air does not affect the flow oftest solution through the device or prevent the test solution fromreaching the reaction well.

The housing preferably has two parts which are capable of moving withrespect to each other while remaining connected to one another. Theaction of moving the parts may provide the driving mechanism by whichdiluent is moved through the device. The diluent can be driven throughthe device by compression of the diluent chamber which forces thediluent past the sample tip and to the reaction well or wells. Thecompression of the diluent chamber can occur when the parts of thehousing are moved with respect to one another such as by sliding onepart past another.

In some embodiments, the housing comprises a locking mechanism whichlocks the housing in position once the driving mechanism has beenactivated and prevents reuse of the device. In this way it isimmediately apparent that the device has been used and cannot be usedagain. This minimizes false results from, for instance, a device thathas been mistakenly activated in transit or from reuse of a device whosereagents have been spent.

In some embodiments, the sampling component preferably comprises ahandle and a sampling tip, the handle preferably comprising a seal whichengages with the opening in the housing to seal the tube when thesampling component is fully inserted in the tube. The seal generallyprevents escape of the sample and diluent from the device reducing thechance of cross contamination from the wound fluid. Preferably the sealand tube engage to lock the sampling component in the device and preventremoval of the sampling component once it has been used. This furtherreduces the chance of cross contamination from the sampling component.

Preferably insertion of the sampling component in the device releasesthe seal on the diluent chamber. Preferably the seal is a ball valve orcan be a film or membrane seal or a duck bill valve or other non-returnvalve known in the art which is activated when the sampling component isinserted in the device. The sampling component preferably bursts,punctures or displaces the seal on the diluent chamber when it isinserted in the device.

Preferably the tube is the same or similar size to the sampling tip ofthe sampling component so that the act of inserting the sampling tipinto the tube causes it to be scraped along the walls of the tube aidingthe dispersion of the sample in the diluent once it is released from thediluent chamber and is flushed through the device. The sizing of thesampling tip to match the tube also forces the diluent to be flushedthrough the tip when the diluent is driven from the diluent chamber.Preferably the diluent chamber is shaped like a bellows to assist in thecompression of the chamber. Alternatively the chamber can be acombination of a plunger and tube similar to that found in a syringe orcan be a filled flexible sachet which is compressed by hand by the useror a balloon which contracts when the seal is released.

In some embodiments, the kit comprises a sampling component forcollecting a sample and a test device. The test device comprises ahousing surrounding a tube to define an opening the housing to receivethe sampling component, the housing having within it a sealed diluentchamber which is connected to an opposite end of the tube and holding aliquid diluent for removing the sample from the sampling tip to form atest liquid. The tube is in liquid communication with a reaction wellwhich holds a reagent capable of indicating the presence of an analyte.

A driving mechanism drives the diluent from the chamber past thesampling tip, into the tube and finally to the reaction well.

FIG. 26 shows a cross section of a standalone device kit for detectingan analyte in a sample. The sampling component (2) comprises a handle(4) and a sampling tip (6) in the process of being inserted into thehousing through one end of a tube (10). The sampling component (2) has asealing means (12) which forms a seal with the open end of the tube (10)while the sampling tip (6) depresses the ball valve (14) to open thediluent chamber (16). FIG. 27 shows a sampling tip fully inserted in thehousing to seal the component to the device. FIG. 28 shows a plan viewof the standalone device kit with the sampling component in place andshows three viewing windows (20) to the left of the housing whichcoincide with three reaction chambers (18) which contain a reagentcapable of indicating the presence of an analyte. The reaction chambersmay contain reagents capable of detecting different analytes from forinstance a wound fluid. The window on the right of the housing whenviewed from above is a control window which indicates that the test hastaken place. Housing (8) is in two main parts which are slidablyconnected to each other. In FIG. 29, a user of the device can slide alower part of the housing (24) away from the upper part of the housing(26) and in so doing cause a lever (28) to compress the diluent chamber(16) and drive the diluent out of the chamber, through the sampling tip(6) and up tube (10) to manifold (30). A plan view (FIG. 30) of thestandalone device kit with housing slid apart, which results in windows(20) and control window (22) indicating that a test has taken place. Thearrows (A) in FIG. 29 indicate the movement of the diluent through thedevice to form a test solution. Diluent chamber, tube and reactionchamber in the standalone device kit are shown in FIG. 31, with thehousing removed for clarity. FIG. 32 shows distribution of test solutionto each reaction chamber in a standalone device kit. Test solution flowsto each reaction chamber (18) from a central node (32). The node (32)may also contain a non-return valve to prevent test solution fromflowing back into the device and causing cross contamination.

The sampling component comprises a handle and a sampling tip in theprocess of being inserted into the housing through one end of a tube.The sampling component has a sealing means which forms a seal with theopen end of the tube while the sampling tip depresses the ball valve toopen the diluent chamber. The sampling tip, when fully inserted in thehousing to seal the component to the device, allows the housing to beopened, releasing the diluent and allowing the forcing means to operate.

The device also comprises three viewing windows in the housing thatcorrespond to three reaction chambers which contain a reagent capable ofindicating the presence of an analyte. The reaction chambers may containreagents capable of detecting different analytes from for instance awound fluid. Some embodiments include a control window which indicatesthat the test has taken place and that the sample was sufficient to makethe test viable.

The user of the device can slide a lower part of the housing away fromthe upper part of the housing and, in so doing, cause a lever tocompress the diluent chamber and drive the diluent out of the chamber,through the sampling tip and up tube to manifold. If the device is notactivated, that is if the seal on the diluent chamber has not beenbroken, it is not possible for the housing to open. The opening of thehousing causes the viewing windows to be positioned over the reactionwells and enable the result to be viewed by the user. This provides asafety measure as it ensures that proper operation of the device inorder to obtain a reliable result.

Once activated, the test solution flows to each reaction chamber from acentral node. In some embodiments, the node comprises a non-return valveand filter to prevent test solution from flowing back into the deviceand between reaction chambers, which can cause cross contamination. Thepathway for the flow of diluent through the device is preferablyprovided with vents at the reaction chamber end.

Example 17. Devices with Separate Sample Preparation Chamber

FIG. 33 shows a diagnostic swab device with housing. In one embodimentthe swab device comprises a resealable housing (80), further comprisinglocator and locking pins (82), a viewing window (81) for observingvisible signals from reagent disks placed in disk holders (83), and agroove (85) for placing the swab. Side view of FIG. 33(C) shows thehousing (80). To use, a user touches a sample with the swab, places theswab in the housing (80) in groove (85), pull on the stem of the swab asshown by the arrow in (D) so that the sample on the swab slides on thestrip (86) and transfers the sample to reagent or indicator disks (83).The results may be viewed through viewing window (81). The swab may alsobe kept in the housing (80) for analysis later.

FIG. 34 shows one embodiment of a thread hook sample preparation device(200), comprising a needle-like tip and a handle or plunger (201),wherein the tip further comprises a hook for extracting a thread from adressing without disturbing the dressing as shown in FIG. 34(A). Uponextracting a thread from the dressing, thread hook device (200) may beinserted into a sample preparation chamber or diluent chamber (202)containing a diluent for dissolving or diluting microbial biomarkers orwound fluid from the thread FIGS. 34(B) and 34(C). The plunger (201) ofthe thread hook device may be depressed downward in the samplepreparation chamber (202) so that the tip of the needle breaks a seal asshown in FIG. 34(D) at the bottom of the sample preparation chamber(203) in order to release the sample solution into a device for analysisof wound fluid or surgical site.

FIG. 35 shows one embodiment of a swab sample preparation device (300),comprising a swab (302) with a handle or plunger (301) may be used totouch a sample for testing. The swab device (300), after sampling abodily fluid or wound fluid, is placed inside a sample preparationchamber (202) containing a buffer for dissolving or diluting the woundfluid or bodily fluid as seen in FIG. 35(A). The swab device is agitatedor mixed inside the sample preparation chamber to further release thefluid sample into the sample preparation chamber as shown in FIG. 35(B).The plunger (301) of the needle is depressed downward as shown in FIG.35(C) to break the seal (203) at the bottom of the sample preparationchamber, allowing the sample fluid to flow into a reaction chambercontaining reagents or indicator inserts or disks for detectingmicrobial infection in the sample taken by the swab. In some embodimentsas shown in FIG. 35(D), gas is removed using Goretex membranes (204)which are gas and vapor permeable, but not permeable to liquid water.Said membranes can be used to degas both the sample as it is injectedand to vent the fluid chambers where the assay takes place.

FIG. 36 shows a sample preparation chamber adapted to indicator testing.Sample preparation chamber (202) is adapted for dissolving or diluting asample for testing further and comprises a resealable top (401) and abreakable seal (402) at the bottom of the chamber (203), where thesample preparation chamber connects to a reaction chamber or diagnosticdevice. When a swab device or a thread hook device is plunged downwardor depressed downward in the chamber, it causes the seal (402) at thebottom to break, releasing sample fluid into a diagnostic deviceconnected to the chamber.

In a further embodiment, FIG. 37, a diagnostic device (500) or analysissystem is adapted to connecting to the sample preparation chamber (202)at one end, allowing sample fluid to flow from the sample tip (300) uponbreaking the seal (203) at the chamber connector, which allows thesample fluid to flow from the sample preparation chamber (202) intoreaction chambers (502) for analysis. Absorbent material (501)positioned after the reaction chambers (502) helps to draw the samplefluid from the sample preparation chamber (202) into the reactionchambers (502). Reaction chambers may contain reagents, reagent tablets,reagent disks, or indicator inserts, as described herein.

In so far as liquid phase tests are desired, they may be conducted usinga variety of means but ultimately rely on the formation of a visiblesignal in a low volume of liquid (e.g. 100 μL). The methods differ interms of how one acquires, dilutes and introduces the sample. In thisexample, we introduce the sample using an adapted syringe-likeconfiguration. The sample may be a swab, piece of gauze or contaminatedthread from a dressing. The swab (FIG. 35) is placed in a plungerconfiguration and then the plunger forms a handle with which the swabcan be mixed with an extraction buffer or a diluent in a samplepreparation chamber. The plunger then allows the removal of fluid bysealing against the stem of the swab and the sides of the chambersimultaneously; a goretex insert in the plunger allows gas removal asthe plunger descends. Where the sample is a thread or piece of gauze,the swab is replaced by a hook, however, the principle is the same asthe stem of the hook is placed within the plunger.

The sample preparation chamber contains buffer which is mixed with thesample on the swab/hook. The chamber is sealed at the Luer-Lock styleconnector and this seal is broken either when the Luer is placed in areceptacle, or when the swab or hook is pushed through the bottom of thechamber (FIG. 35). The assay device entrance includes a standard femaleLuer with a Luer lock like surround to ensure good sealing. The modifiedchamber engages irreversibly with the female Luer lock and on depressionof the plunger, the fluid is transferred gas free into the device via afluid distribution network. Each chamber in the device contains areagent tablet (see previous example for the reagents). Each chamber isvented via a goretex patch sonic welded over the chamber. As soon as thechamber is filled (from bottom to top), fluid flow preferably stops. Thevented gas passes by a filter before reaching the atmosphere. Thearrival of fluid dissolves the reagent pills and allows the reaction tostart. The degree of reaction over a given time is determined bycomparison to a chart of colors. The result is largely binary, clearcolor or not. The more markers associated with color, the more likelythe potential infection. Thus, wound fluids from uninfected wounds donot cause color change. Those from infected wounds cause at least onemarker to change color and more often all three markers within 5minutes.

FIG. 38 shows one embodiment of a diagnostic device or a transfersystem, comprising a chamber or vessel (601) containing a buffer, suchas saline, a resealable top (600), a plunger or similar device (602)with a gas outlet and a hook or sample tip at one end for transferringsample into the a sample preparation chamber or a diluent chamber (601),and at least one reaction chamber (606) capable of analyzing a samplefluid from the chamber (601). To conduct such analysis, the plunger orpiston (602) containing a sample at the end is inserted into the samplepreparation chamber (601), or a sample is placed in the diluent chamber(601), so that the sample may be diluted or dissolved in the buffer inthe chamber (601). The assembly of a plunger (602) inserted in a diluentchamber (601) is shown in (607).

The chamber (601) may further comprise a Luer-Lock or slip tip (605) forconnecting to a reaction chamber (604) or an analysis system. Afterconnecting plunger unit (601, 602) to reaction chamber (604), one maydepress the plunger (602) downward to break a seal at the end of thechamber (601), releasing sample fluid from the sample preparationchamber into reaction chambers (604), wherein individual reactionchamber (606) may have a different reporter or color system fordetecting an analyte. The plunger (602) can further comprise membranethat pushes water and lets out gas, thus degassing the sample fluid asone depresses the plunger into the chamber. The reaction chambers may befilled in parallel, and the last chamber contains an aerosol filter anda pressure exit to atmosphere. Pressure, equalization, reaction chamberfilling and aerosol filtering can be achieved through membrane exits. Insome embodiments, reaction chambers contain reagent tablets or reagentdisks. Top membranes can be welded in place using ultrasound. Lensesthat enlarge the view of the reaction chambers are used in someembodiments. The connection to the reaction chamber or transfer system(604) includes a rough filter and a penetrator for breaking the bufferseal on connection at 605. Reaction chambers can be closed at the topand bottom by clipping on.

The conformation of the reaction vessels can be flexibly organized. Oneexample is shown in FIG. 39, which shows another embodiment of ananalysis system (604). Reaction chambers (606) can be arranged in aradial manner instead of in a linear arrangement. A fan- orradial-shaped analysis system (604) is adapted to use with a samplepreparation chamber (601) with a plunger (602) system for driving asample solution into reaction wells or chambers. Different views of suchanalysis system (604) are shown in (B). (608) shows a top view of aseries of reaction chambers arranged in a radial arrangement. In someembodiments, the reaction chamber unit (610) may be removable fromhousing (609). This removable feature facilitates a user in refilling,inserting, or exchanging reagents in individual reaction chambers withinthe reaction unit (610).

In this example, the reagents used are water soluble and are formulatedas tablets using excipients such as PEG, maltose and sorbitol ascarriers. The tablets are formulated with the appropriate amounts ofbuffer salts in the bulk mixture to result in optimal pH upondissolution. For supply of hydrogen peroxide, sodium percarbonate isused. As an MPO substrate, a soluble Fast Blue derivative, i.e theproduct of reaction with succinic anhydrice, is used, alternatively,guacol, diamino phenol or similar may be used. For Elastase, AAPVnitrophenol amide is employed, alternatively, AAPV-indoxyl with adiazonium salt enhancer. For Lysozyme, the substrate is a labelledpeptidoglycan particle, however, the well contains a positively chargedmembrane at the viewing interface. This membrane is derived on one halfwith the trap, and the contrast between the two sides in the mainindicator of reaction indicates the degree of reaction.

In some embodiments, such as FIG. 24, sampling threads (100) are builtin or added to dressing for a wound or at a surgical site (92). AQUACEL(4) is used in some embodiments of the dressing (92). Sampling threadsabsorb wound fluid or fluid at surgical site (D). A thread may be pulledout or extracted as shown in FIG. 24(E) from dressing without having toremove or disturb dressing using an instrument (101) such as a tweezer,hook, or thread hook device. The thread can then be dissolved in abuffer for use in a diagnostic device (102) using one or more indicatorregents or indicator disks described herein.

In some embodiments, a wound dressing comprises built in samplingthreads. In some embodiments, the sampling threads absorb wound fluidsand may be removed without disturbing the wound dressing for detectionof analytes in the wound fluid.

In some embodiments, the sample threads may be diluted in buffer todissolve markers for diagnosing the status of the surgical site orwound.

In some embodiments, a thread hook device may be used to remove a threadfrom a wound dressing.

While preferred embodiments of the disclosed technology have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosed technology. It should beunderstood that various alternatives to the embodiments of the disclosedtechnology described herein may be employed in practicing the disclosedtechnology. It is intended that the following claims define the scope ofthe disclosed technology and that methods and structures within thescope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A wound dressing comprising a) a wound contactinglayer; b) a reagent layer comprising one or more testing regions,wherein the reagent layer is in fluid communication with the woundcontacting layer; and c) an outer layer that overlays the reagent layer.2. The wound dressing of claim 1, wherein the wound contacting layercomprises gel-forming polymers.
 3. The wound dressing of claim 1,wherein each of the one or more testing regions comprises one or more ofeach of a back-flow trap, a reagent pad, a filter pad, an indicatortrap, and an absorbent area, and wherein one or more viewing windows arelocated either above the reagent pad or the indicator trap.
 4. The wounddressing of claim 3, wherein: a) the reagent pad is in fluidcommunication with the filter pad; b) the filter pad is in fluidcommunication with the indicator trap; and c) the indicator trap is influid communication with the absorbent area.
 5. The wound dressing ofclaim 1, wherein each of the one or more testing regions comprises oneor more reagents selected from the group consisting of enzyme-reactiveindicators, reagents that are sources of peroxide, enzymes that producecolored products, pH indicators, protein responsive reagents, andmoisture-detecting reagents.
 6. The wound dressing of claim 5, whereinenzyme-reactive indicators are protein-indicator conjugates.
 7. Thewound dressing of claim 6, wherein the protein-indicator conjugates aredeposited in or on the reagent pad.
 8. The wound dressing of claim 6,wherein the protein-indicator conjugate has the structure of Formula(I):A-B   Formula (I) wherein: A is an anchor region or moiety that helps tobind an enzyme-reactive region to the reagent pad; and B is theenzyme-reactive region.
 9. The wound dressing of claim 8, wherein theenzyme-reactive region comprises a peptide.
 10. The wound dressing ofclaim 8, wherein the enzyme-reactive region comprises an indicatorregion having an enzyme-reaction indicator.
 11. The wound dressing ofclaim 8, wherein B further comprises an indicator region.
 12. The wounddressing of claim 11, wherein the indicator region, after having beencleaved by the target enzyme is transformed into a colored species byaccessory enzymes selected from the group consisting of lipase,esterase, hexosaminidase, peroxidase, oxidase, glycosidase, glucosidase,laccase, and a combination of two or more thereof.
 13. The wounddressing of claim 10, wherein the enzyme-reactive indicators interactwith one or more enzymes selected from the group consisting of elastase,lysozyme, cathepsin G, myeloperoxidase, and any combination thereof. 14.The wound dressing of claim 10, wherein the enzyme-reactive indicatorscomprise a moiety capable of producing a visible color or a detectableelectronic change upon interaction of the enzyme-labile orenzyme-reactive region with one or more enzymes, wherein the moiety isselected from the group consisting of a peroxidase substrate, arylamine,an amino phenol, a neutral dye, a charged dye, a nanoparticle, acolloidal gold particle, and an analog thereof.
 15. The wound dressingof claim 8, wherein the anchor region is covalently attached to thereagent pad.
 16. The wound dressing of claim 8, wherein the anchorregion is non-covalently attached to the reagent pad.
 17. The wounddressing of claim 16, wherein the anchor region is ionically attached tothe reagent pad.
 18. The wound dressing of claim 1, further comprisingone or more lines of wicking stitching or wicking tufting throughout alllayers of the wound dressing except the outer layer, and wherein thewicking stitching or wicking tufting provides fluid communicationbetween the reagent layer and the wound contacting layer.
 19. The wounddressing of claim 18, wherein one or more lines of wicking stitching orwicking tufting comprise fibers that are wettable and exhibit capillaryaction.
 20. The wound dressing of claim 19, wherein the fibers comprisecotton, rayon, viscose, wool, silk, polyester, polyamide, CMC,polypropylene, or any combination thereof.
 21. The wound dressing ofclaim 1, wherein one or more testing regions comprise a leach-back trapin fluid communication with a reagent pad and one or more lines ofwicking stitching or wicking tufting crossing through one or moretesting regions only at the leach-back trap.
 22. The wound dressing ofclaim 1, further comprising a foam layer between the wound contactinglayer and the reagent layer.
 23. The wound dressing of claim 22, furthercomprising one or more perforations in the wound contacting layer. 24.The wound dressing of claim 22, further comprising one or moreperforations in the foam layer and the wound contacting layer.
 25. Thewound dressing of claim 21, wherein each of the one or more testingregions further comprises a leach-back trap in fluid communication withthe reagent pad and one or more perforations aligned with the leach-backtrap.
 26. The wound dressing of claim 1, wherein each of the one or moretesting regions comprises a multichannel testing region, wherein eachchannel is separated from an adjacent channel by one or more impermeableseparators or borders.
 27. The wound dressing of claim 26, comprising aplurality of testing regions.
 28. The wound dressing of claims 26,wherein the testing regions are arranged in a linear configuration. 29.The wound dressing of claim 26, wherein the testing regions are arrangedin a radial configuration.
 30. The wound dressing of claim 1, whereinthe outer layer has one or more windows that permit visualization of asignal from the reagent layer.
 31. The wound dressing of claim 30,wherein the signal is a color change.
 32. The wound dressing of claim30, wherein the signal is a fluorescent signal, a luminescent signal, ora signal mediated by physical means, such as an electrical change.
 33. Amethod of detecting the level of one or more enzymes in a mammalianwound, the method comprising: a) contacting the mammalian wound with awound dressing of claim 1; b) observing one or more signals in thereagent layer, wherein the signal is a color change, a fluorescentsignal, a luminescent signal, or an electrical change; and c) comparingthe signal to a reference or a control to determine the level of anenzyme.
 34. A method of detecting the presence of one or more enzymes ina mammalian wound, the method comprising: a) contacting the mammalianwound with a wound dressing of claim 1; and b) observing one or moresignals in the reagent layer, wherein the signal is a color change, afluorescent signal, a luminescent signal, or an electrical change.
 35. Amethod of detecting an infection in a mammalian wound, the methodcomprising: a) contacting the wound with a wound dressing of claim 1;and b) observing one or more signals in the reagent layer, wherein thesignal is a color change, a fluorescent signal, a luminescent signal, oran electrical change.
 36. A method of treating an infection in a woundof a mammal, the method comprising: a) contacting the wound with a wounddressing of claim 1; b) observing one or more signals in the reagentlayer indicative of an infection, wherein the signal is a color change,a fluorescent signal, a luminescent signal, or an electrical change; andc) administering a medical treatment to the mammal.
 37. A device fordetecting an infection in a wound, comprising: a) a wound contactinglayer; b) a reaction layer comprising one or more reagents that canindicate the presence of one or more analytes associated with aninfection, wherein the reagents are affixed to a solid phase and producea detectable signal in a reporter area; c) a cover on top of thereaction layer, wherein the cover comprises one or more windows or clearareas to allow visualization of the detectable signal; and d) fluidcommunication between the wound contacting layer and the reaction layer.38. The device of claim 37, wherein the reagents compriseenzyme-reactive regions that interact with one or more target enzymesselected from the group consisting of lysozyme, MPO, cathepsin G,elastase, catalase, lipase, esterase, and any combination thereof. 39.The device of claim 37, wherein one or more reagents produce a visiblecolor upon a change in the pH, at an acidic pH, or at a basic pH, andwherein the pH-sensitive reagent comprises bromothymol blue, phenol red,bromophenol red, chlorophenol red, thymol blue, bromocresol green,bromocresol purple; nitrazine yellow; or other sulfophthalein dyes. 40.The device of claim 38, wherein the enzyme-reactive regions comprise amoiety capable of producing a visible color or detectable electronicchange upon interaction with one or more target enzymes, wherein themoiety is a peroxidase substrate, arylamine, an amino phenol, anindoxyl, a neutral dye, a charged dye, a nanoparticle, a colloidal goldparticle, or an analog thereof.
 41. The device of claim 40, wherein thereagent interacts with a target enzyme to produce a colored species orto produce an intermediate product that interacts with an accessoryenzyme selected from the group consisting of a lipase, esterase,hexosaminidase, peroxidase, oxidase, glycosidase, glucosidase, andlaccase.
 42. The device of claim 37, wherein the fluid communicationcomprises wicking stitching or wicking tufting of an absorbent materialthat allows fluid communication between the wound contacting layer andthe reaction layer.
 43. The device of claim 37, wherein the reagents areprinted, sprayed, or deposited on the solid phase.
 44. The device ofclaim 37, wherein the solid phase is selected from the group consistingof paper, viscose, regenerated cellulose, glass fiber, and anycombination thereof.
 45. The device of claim 37, wherein the detectablesignal is a color change, a fluorescent signal, a luminescent signal, oran electrical change.
 46. The device of claim 37, wherein the device isa wound dressing.
 47. A device for detection of infection associatedenzymes that is provided as an independent entity and can be placed inany dressing system, comprising a sample inlet in fluid communicationwith reagent cells, wherein reagent cells comprise indicators for sampledelivery and/or pH, and one or more indicators for biomarkers of aninfection selected from the group consisting of lysozyme, MPO, cathepsinG, elastase, catalase, lipase, and esterase.
 48. The device of claim 47,wherein the indicators comprise enzyme-reactive indicators, reagentsthat are sources of peroxide, enzymes that produce colored products, pHindicators, protein responsive reagents, or moisture-detecting reagents.49. The device of claim 47, wherein the indicators are enzyme-reactiveindicators or protein-indicator conjugates.
 50. The device of claim 47,wherein the protein-indicator conjugates are deposited in or on thereagent pad.
 51. The device of claim 47, wherein the protein-indicatorconjugate has the structure of Formula (I): A-B, wherein A is an anchorregion or moiety that helps to bind an enzyme-reactive region to thereagent pad; B is the enzyme-reactive region; and wherein theenzyme-reactive region comprises a peptide or an indicator region. 52.The device of claim 51, wherein the indicator region interacts with anenzyme to produce a colored species or wherein an intermediate speciesinteracts with an accessory enzyme selected from a group consisting of alipase, esterase, hexosaminidase, peroxidase, oxidase, glycosidase,glucosidase, and laccase to produce a colored species.
 53. The device ofclaim 47, wherein the fluid communication comprises one to ten indicatorchannels separated by impermeable lanes, borders, or separators, whereineach indicator channel comprises a different reagent or control.
 54. Thedevice of claim 47, wherein the fluid communication comprises aplurality of separate indicator channels, and wherein each indicatorchannel comprises a different reagent or control.
 55. The device ofclaim 47, wherein the reagents are printed, sprayed, or deposited on asolid phase in a radial configuration to form a disk.
 56. The device ofclaim 47, wherein the reagents are printed, sprayed, or deposited on asolid phase in a linear configuration to form a testing strip ordipstick-type device.
 57. The device of claim 55, wherein the diskcomprises reagents printed, sprayed, or deposited on the top surface ofthe disk with a trap material and a substrate material on the bottomsurface, wherein the substrate can be digested by one or more enzymes inthe sample to release one or more products that migrate toward the trap.58. The device of claim 57, wherein one or more products are colored orproduce a color change through interaction with an accessory enzyme, andwherein the color change can be visualized on the top surface of thedisk.
 59. A diagnostic disk for detecting an infection in a wound,comprising: a) a reaction layer comprising one or more reagents thatinteract with a target enzyme indicative of an infection, wherein thereagents are affixed to a solid phase; b) each reagent is sprayed,printed, or deposited in a reagent area in a lane separated fromadjacent lanes by impermeable separators; c) each lane comprises areporter area wherein a color, color change, or other detectable signalis observed; and d) a cover comprising a window for visualizing thesignal in the reporter area.
 60. The diagnostic disk of claim 59,wherein one or more reagents produce a visible color upon a change inthe pH, at an acidic pH, or at a basic pH, wherein the pH-sensitivereagent comprises bromothymol blue, phenol red, bromophenol red,chlorophenol red, thymol blue, bromocresol green, bromocresol purple;nitrazine yellow; or other sulfophthalein dyes.
 61. The diagnostic diskof claim 59, wherein multiple lanes are arranged in a linear or radialconfiguration about a cut access, perforation, or wicking material thatallows fluid communication between a wound contacting area to thereagents in the reaction layer.
 62. The diagnostic disk of claim 59,wherein one or more reagents produce a color signal or other detectablesignal upon interaction with an enzyme selected from the groupconsisting of lysozyme, MPO, cathepsin G, elastase, catalase, lipase,and esterase.
 63. The diagnostic disk of claim 59, wherein the reagentscomprise a moiety selected from the group consisting of peroxidasesubstrate, arylamine, an amino phenol, an indoxyl, a neutral dye, acharged dye, a nanoparticle, and a colloidal gold particle, and ananalog thereof.
 64. The diagnostic disk of claim 59, wherein thereagents comprise accessory enzymes that produce a colored species orcolor signal, and wherein the accessory enzyme is a lipase, esterase,hexosaminidase, peroxidase, oxidase, glycosidase, glucosidase, laccase,or a combination of one or more thereof.
 65. The diagnostic disk ofclaim 59, wherein the solid phase is selected from the group consistingof paper, viscose, regenerated cellulose, glass fiber, and similarmaterial.
 66. The diagnostic disk of claim 59, wherein there are aplurality of lanes, each with a different reagent or control.
 67. Thediagnostic disk of claim 59, wherein the detectable signal comprises acolor change, appearance or disappearance of a color, a fluorescentsignal, a luminescent signal, or an electrical change or signal.
 68. Alateral flow or dipstick device for detecting an infection in a wound,comprising: one or more reagent disks arranged in a linearconfiguration, wherein each reagent disk is impregnated with a reagentthat interacts with an enzyme to produce a color change and/or ispH-sensitive, comprising bromothymol blue, phenol red, bromophenol red,chlorophenol red, thymol blue, bromocresol green, bromocresol purple;nitrazine yellow; or other sulfophthalein dyes, and wherein the disksare affixed to a solid phase.
 69. The device in claim 68, wherein thereagents produce a color signal upon interaction with an enzyme selectedfrom the group consisting of lysozyme, MPO, cathepsin G, elastase,catalase, lipase, and esterase.
 70. The device in claim 68, wherein thesolid phase is selected from the group consisting of paper, viscose,regenerated cellulose, glass fiber, and any combination thereof.
 71. Adevice for detecting an infection in a wound or a sample, comprising ahousing, wherein the housing comprises: a) a sampling component forcollecting the sample; b) a sample preparation chamber in fluidcommunication with a reaction chamber, wherein the sample preparationchamber receives the sample; c) the reaction chamber comprising one ormore reaction cells containing reagents that interact with one or moreenzymes in the sample to indicate the presence of an infection and/or pHof the sample; and d) a window or a clear area for visualizing adetectable signal, wherein the signal is a color change.
 72. The devicein claim 71, wherein one or more reagents produce a color signal uponinteraction with an enzyme selected from a group consisting of lysozyme,MPO, cathepsin G, elastase, catalase, lipase, and esterase.
 73. Thedevice in claim 71, wherein one of the reagents produces a color changein response to a change in pH, a basic pH, or an acidic pH.
 74. Thedevice in claim 71, wherein the reagents perform in a primarily liquidmedium.
 75. The device in claim 71, wherein the reagents are provided intablet form for use in the reaction cells.
 76. The device in claim 71,wherein the reagents are printed, sprayed, or deposited in separatereagent fields on a support material to form a panel of tests for use inthe reaction chamber.
 77. The device in 71, wherein the support materialis selected from the group consisting of paper, viscose, regeneratedcellulose, and glass fiber, arrayed in a line along a carrier strip. 78.The device in claim 76, wherein the reagent fields are arrayed in a linealong a carrier strip capable of absorbing the sample in the reactionchamber.
 79. The device in claim 71, wherein the sampling component is aswab device.
 80. The device in claim 71, wherein the sampling componentis a hook or needle device adapted to removing a thread from a wounddressing without disturbing the wound dressing.
 81. A kit for detectingan infection in a sample, comprising: a) a sampling component forcollecting the sample; b) a test device comprising a housing surroundinga tube to define an opening in the housing for receiving the samplingcomponent, the housing comprising: c) a diluent chamber that holds aliquid diluent; d) a reaction well in liquid communication with thetube, wherein the reaction well holds one or more reagents that interactwith one or more analytes to produce a color change or a detectablesignal; e) a viewing window or reporter area wherein the color change ordetectable signal can be observed; and f) wherein the liquid diluentflows from the sample component into the reaction well to mix the samplewith the reagents in the reaction well.
 82. The kit of claim 81, whereinthe reagents comprise one or more enzyme-reactive indicators and/or pHindicator.
 83. The kit of claim 81, wherein one or more reagents producea color signal upon interaction with an enzyme selected from a groupconsisting of lysozyme, MPO, cathepsin G, elastase, catalase, lipase,and esterase.
 84. The kit of claim 81, wherein the reagents comprise amoiety selected from the group consisting of peroxidase substrate,arylamine, an amino phenol, an indoxyl, a neutral dye, a charged dye, ananoparticle, a colloidal gold particle, and an analog thereof.
 85. Thekit of claim 81, wherein the detectable signal comprises a color signalor color change, a fluorescent signal, a luminescent signal, or anelectrical signal.
 86. The kit of claim 81, wherein at least one reagentproduces a color signal in response to a basic pH, an acidic pH, or achange in pH, wherein the pH-sensitive reagent is bromothymol blue,phenol red, bromophenol red, chlorophenol red, thymol blue, bromocresolgreen, bromocresol purple; nitrazine yellow; or other sulfophthaleindyes.
 87. The kit of claim 81, wherein the sample is obtained from awound, a wound dressing, or a surgical site.
 88. The kit of claim 81,wherein the sampling component is a swab device or a hook or needledevice.
 89. The kit of claim 81, wherein the reagents are deposited inseparate fields on a testing strip to form a panel of tests.
 90. The kitof claim 81, wherein there are a plurality of reaction wells, whereineach reaction well comprises a different reagent or control.
 91. The kitof claim 90, wherein the reaction wells are arranged in a linearconfiguration.
 92. The kit of claim 90, wherein the reaction wells arearranged in a radial configuration.